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Dempsey, Nunez Laura. "Spectrum of mutations in MMAA identified by high resolution melting analysis". Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=110535.
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The gene product of MMAA is required for the intracellular metabolism of cobalamin (Cbl). Mutations in this gene lead to the cblA class of disorders, characterized by isolated methylmalonic aciduria. We have been concerned that somatic cell methods of diagnosis may miss patients with mild cellular phenotypes. A high resolution melting (HRM) analysis assay was developed to rapidly scan the coding exons and flanking intronic regions of the MMAA genes for variants. DNA from 96 unaffected reference individuals, 72 patients with complementation confirmed cblA, and 181 patients with elevated isolated methylmalonic acid, who could not be diagnosed using complementation analysis, were scanned by HRM. Suspected variants were confirmed using Sanger sequencing. In the cblA cohort, HRM correctly identified all previously known mutations as well as an additional 22 variants, 10 of which had not been previously reported. Novel variants included one duplication (C.551dupG, p.C187LfsX3), one deletion (c.387delC, p.Y129YfsX13), one splice site mutation (c.440-2A>G, splice site), 4 missense mutations (c.748G>A, p.E520K; c.820G>A, p.G274S; c.627G>T, p.R209S; c.826A>G, p.K276E), and 3 nonsense mutations (c.960G>A, p.W320X; c.1075C>T, p.E359X; c.1084C>T, p.Q362X). All novel missense variants, listed above, affect highly conserved residues and are predicted to be damaging. Scanning of MMAA in the 181 undiagnosed samples revealed a single novel heterozygous missense change (c.821G>A, p.G274D).Le produit génique du MMAA est nécessaire pour le métabolisme de la cobalamine intracellulaire (Cbl). Des mutations dans ce gène conduisent à la classe de maladies cblA, caractérisé par l'acidurie méthylmalonique isolée. Nous avons été concernés que les méthodes de diagnostic de cellules somatiques peuvent manquer les patients atteints phénotypes cellulaires moins sévère. Une teste de fusion à haute résolution a été développé pour balayer rapidement les exons codantes et les régions introniques adjacentes du gène MMAA pour des variantes. Nous avons testé l'ADN à partir de 96 personnes de référence qui ne sont pas touchés, 72 patients atteints de cblA confirmé par complémentation et 181 patients présentant une élévation de l'acide méthylmalonique isolée, qui ne pouvaient pas être diagnostiquée à l'aide d'analyse de complémentation. Les variantes suspectes ont été confirmées à l'aide de séquençage Sanger. Dans la cohorte cblA, l'analyse de fusion à haute résolution a correctement identifié toutes les mutations connues antérieurement, ainsi que 22 autres variantes, dont 10 n'avaient pas été signalés précédemment. Nouveaux variantes inclus une duplication (C.551dupG, p.C187LfsX3), une délétion (c.387delC, p.Y129YfsX13), une mutation du site d'épissage (c.440-2A> G, site d'épissage), 4 mutations faux-sens (c. 748G> A, p.E520K; c.820G> A, p.G274S; c.627G> T, p.R209S; c.826A> G, p.K276E), et 3 mutations non-sens (c.960G> A, p.W320X; c.1075C> T, p.E359X; c.1084C> T, p.Q362X). Toutes les variantes faux-sens nouveaux, énumérés ci-dessus, affectent des résidus hautement conservés et sont prévus pour être endommageant. L'analyse de MMAA dans les 181 échantillons non diagnostiqués a révélé un seul changement faux-sens hétérozygote (c.821G> A, p.G274D).
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Illson, Margaret. "Spectrum of mutations in MMAB identified by high resolution melting analysis". Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=110564.
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Pathogenic variants in the MMAB gene (OMIM 607958) are responsible for the cblB class of cobalamin-responsive methylmalonic aciduria (MMA) (OMIM 251110). MMAB encodes cobalamin adenosyltransferase, a mitochondrial enzyme responsible for the formation of adenosylcobalamin (AdoCbl). AdoCbl subsequently functions as a cofactor for methylmalonyl-CoA mutase (MCM) during the isomerization of L-methylmalonyl-CoA to succinyl-CoA. Somatic cells studies have been used to evaluate patient samples for cobalamin related disorders. Due to high basal levels of propionate incorporation, some patients with mild MMA biochemical phenotypes cannot be diagnosed by complementation analysis. A high resolution melting analysis (HRMA) assay was developed to rapidly scan the coding exons and flanking intronic regions for variants in the MMAB gene.Three cohorts of samples were scanned by HRMA: an unaffected reference population, 42 samples assigned to the cblB by complementation analysis and 181 patients with unresolved isolated MMA. HRMA correctly identified all of the previously reported mutations in the cblB cohort as well as seven additional variants including a novel nonsense variant (c.12C>A, p.C4X). Scanning of the unresolved MMA cohort identified six samples containing MMAB variants. Two samples, WG3948 and WG4034, contained compound heterozygous variants. They shared a c.572 G>A (p.R191Q) mutation. WG3948, the index case for this study, was found to have c.398 C>T (p.S133F) as the second mutation, and WG4034, the second patient, contained a novel variant c.394 C>T (p.C132R). Samples from four other affected patients contained a single variant. The c.572 G>A (p. R191Q) was found in both WG3546 and WG4090. WG3759 contained a c.521C>T ( p.S174L) substitution, and WG4029 contained a novel c.185 C>T (p.T62M) substitution. The identification of two patients with compound heterozygous variants in the MMAB gene suggests the existence of an infrequent but distinct atypical cblB phenotype. This subclass is characterized by levels of propionate incorporation and of AdoCbl synthesis within reference ranges, preventing diagnosis by somatic cell studies.Des variantes pathogéniques dans le gène MMAB (OMIM 607958) sont responsables de la classe cblB d'acidurie méthylmalonique (AMM) respondant à la cobalamine (OMIM 251110). MMAB encode cobalamine adénosyltranférase, une enzyme mitochondriale responsable de la formation de l'adénosylcobalamine (AdoCbl). AdoCbl fonctionne par la suite en tant que cofacteur pour méthylmalonyl-CoA mutase (MCM) durant l'isomérisation de L-méthylmalonyl-CoA vers succinyl-CoA. Des analyses sur des cellules somatiques ont été utilisées pour évaluer des échantillons de patients pour des troubles reliés à la cobalamine. En raison de niveaux de base élevés d'incorporation de propionate, certains patients présentant des phénotypes biochimiques bénins d'AMM ne peuvent être diagnostiqués par analyse de complémentation. Une analyse de fusion à haute résolution (AFHR) a été développée pour balayer rapidement les exons codants et les régions introniques avoisinnantes pour des variantes dans le gène MMAB.Trois cohortes d'échantillons ont été balayées par AFHR : une population de référence non-affectée, 42 échantillons assignés au groupe cblB par analyse de complémentation et 181 patients avec une AMM isolée sans diagnostique. L'AFHR a correctement identifié toutes les mutations précédemment rapportées dans la cohorte cblB ainsi que sept variantes additionelles, incluant une nouvelle variante non-sens (c.12C>A, p.C4X). Le balayage de la cohorte avec de l'AMM isolée a identifié six échantillons contenant des variantes dans MMAB. Deux échantillons, WG3948 et WG4034, étaient des porteurs de variantes hétérozygotes composés. Ils partageaient la mutation c.572G>A (p.R191Q). WG3948, le cas index pour cette étude, était porteur du c.398C>T (p.S133F) pour la deuxième mutation et WG4034, le deuxième patient, contenait une nouvel variante c.394C>T (p.C132R). Les échantillons provenant de quatre autres patients atteints contenait une seule variante. Le c.572G>A (p.R191Q) a été trouvé dans WG3546 et WG4090. WG3759 contenait une substitution c.52C>T (p.S174L), et WG4029 contenait une nouvelle substitution c.185C>T (p.T62M).L'identification de deux patients avec des variantes hétérozygotes composées dans le gène MMAB suggère l'existence d'un phénotype rare mais distinct de cblB. Cette sous-classe est charactérisée par des niveaux d'incorporation de propionate et de synthèse d'AdoCbl dans les valeurs normales, empêchant le diagnostique par analyse des cellules somatiques.
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Souza, Roberto Antonio de. "Genotipagem de linhagens de Yersinia spp. por high-resolution melting analysis". Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/60/60135/tde-27062013-151724/.
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O gênero Yersinia pertence à família Enterobacteriaceae e compreende 17 espécies. Y. pestis, Y. pseudotuberculosis e Y. enterocolitica são reconhecidamente patógenos de humanos e animais. Y. pestis cause a peste. Y. pseudotuberculosis e Y. enterocolitica são agentes causadores, sobretudo, de gastroenterites transmitidas por água e alimentos. As demais 14 espécies são, usualmente, consideradas não-patogênicas, com exceção de Y. ruckeri sorogrupo O:1 que causa infecções em peixes. Nas últimas décadas, a tipagem molecular tornou-se uma importante ferramenta nos estudos filogenéticos de numerosos micro-organismos e o desenvolvimento de sistemas de tipagem rápidos e baratos pode facilitar os estudos epidemiológicos de infecções bacterianas. No presente estudo objetivou-se desenvolver um método de genotipagem de Yersinia spp. baseado em high-resolution melting analysis (HRMA) para diferenciar os single-nucleotide polymorphisms (SNPs) presentes nas sequências dos genes 16S rRNA, glnA, gyrB, hsp60 e recA e aplicá-lo na tipagem de 40 linhagens de Y. pseudotuberculosis e 50 linhagens de Y. enterocolitica, bem como separar por HRMA as espécies Y. pseudotuberculosis e Y. enterocolitica. Os SNPs foram determinados nas sequências dos loci acima citados a partir de um conjunto de 119 linhagens de Yersinia spp. depositadas no GenBank/EMBL/DDBJ. Foram encontrados nas sequências dos genes analisados de Y. pseudotuberculosis, Y. enterocolitica, Y. bercovieri, Y. rohdei, Y. intermedia, Y. mollaretii e Y. ruckeri 10, 10, 9, 6, 4, 1 e 1 SNPs, respectivamente. Nenhum SNP foi encontrado nas sequências analisadas de Y. pestis e um grande número de SNPs foi encontrado nas sequências analisadas de Y. frederiksenii, Y. kristensenii e Y. massiliensis, o que impossibilitou a genotipagem dessas espécies por HRMA. As demais espécies não foram analisadas. Foram desenhados pares de primers para flanquear os SNPs encontrados em cada espécie de Yersinia testada. Usando um conjunto de primers espécie-específicos, a diversidade genética de cada espécie de Yersinia foi determinada por HRMA e a análise filogenética foi baseada na sequência concatenada composta pelos nucleotídeos identificados em cada fragmento analisado. O agrupamento foi realizado com o software BioNumerics usando o método UPGMA com 1.000 replicatas de bootstrap. A árvore filogenética ii construída para Y. pseudotuberculosis agrupou as linhagens em clusters bio-sorogrupo específicos. As linhagens do bio-sorogrupo 1/O:1 foram agrupadas em um cluster e as linhagens do bio-sorogrupo 2/O:3 em outro. A árvore filogenética construída para Y. enterocolitica agrupou as linhagens em três grupos. As linhagens altamente patogênicas, do biotipo 1B, foram agrupadas em um cluster, as linhagens de média patogenicidade, dos biotipos 2, 3, 4 e 5, foram agrupadas em um segundo cluster e as linhagens consideradas nãopatogênicas, do biotipo 1A, foram agrupadas em um terceiro cluster. O agrupamento encontrado em Y. pseudotuberculosis e Y. enterocolitica foi consistente com o perfil patogênico característico dessas duas espécies. Nenhuma correlação epidemiológica significativa foi encontrada no agrupamento de Y. bercovieri, Y. rohdei, Y. intermedia, Y. mollaretii e Y. ruckeri de acordo com os resultados de HRMA. Ademais, o método de HRMA aqui desenvolvido foi capaz de separar as espécies Y. pseudotuberculosis e Y. enterocolitica. O método de HRMA desenvolvido nesse estudo pode ser usado como uma alternativa para a genotipagem e para a diferenciação de Y. pseudotuberculosis de Y. enterocolitica. Esse método também pode complementar os métodos baseados em sequências e facilitar os estudos epidemiológicos dessas duas espécies de Yersinia.The genus Yersinia belongs to the family Enterobacteriaceae and comprises 17 species. Y. pestis, Y. pseudotuberculosis and Y. enterocolitica are well recognized human and animal pathogens. Y. pestis causes plague. Y. pseudotuberculosis and Y. enterocolitica are, usually, causative agents of food-waterborne gastroenteritis. The other 14 Yersinia species are considered to be non-pathogenic, with the exception of Y. ruckeri serogroup O:1 which causes infections in fishes. In the last few decades, molecular typing has become an important tool in phylogenetic studies of several microorganisms and the development of fast and inexpensive typing systems can facilitate epidemiological studies of bacterial infections. The present study aimed to develop a method of Yersinia spp. genotyping based on high-resolution melting analysis (HRMA) in order to differentiate the single-nucleotide polymorphisms (SNPs) present in the 16S rRNA, glnA, gyrB, hsp60 and recA sequences and apply it in the typing of 40 Y. pseudotuberculosis strains and 50 Y. enterocolitica strains, as well as, to separate by HRMA the Y. pseudotuberculosis and Y. enterocolitica species. The SNPs were determined in the sequences of the aforementioned loci using a set of 119 Yersinia strains deposited in the GenBank/EMBL/DDBJ database. It were found in the gene sequences analyzed of Y. pseudotuberculosis, Y. enterocolitica, Y. bercovieri, Y. rohdei, Y. intermedia, Y. mollaretii and Y. ruckeri 10, 10, 9, 6, 4, 1 and 1 SNPs, respectively. No SNPs was found in the analyzed sequences of Y. pestis and a large number of SNPs were found in the analyzed sequences of Y. frederiksenii, Y. kristensenii and Y. massiliensis what prevented their genotyping by HRMA. The remaining Yersinia species were not analyzed. It was designed primer pairs to flank the SNPs found in each Yersinia species tested. Using a specie-specific set of primers, the genetic diversity of each Yersinia species used was determined by HRMA and the phylogenetic analysis was based on the concatenated sequence composed by the nucleotides identified in each fragment analyzed. Clustering was performed with the software package BioNumerics using UPGMA method and 1,000 bootstrap replicates. The phylogenetic tree constructed for Y. pseudotuberculosis grouped the strains into bio-serogroups specific clusters. The strains of 1/O:1 bio-serogroup were grouped into one cluster and the strains of 2/O:3 bio-serogroup into iv other cluster. The phylogenetic tree constructed for Y. enterocolitica grouped the strains in three clusters. The highly pathogenic strains, of biotype 1B, were grouped into one cluster, the moderate pathogenic strains, of biotypes 2, 3, 4 and 5, were grouped into a second cluster and, the non-pathogenic strains, of biotype 1A, were grouped into a third cluster. The clusterization of Y. pseudotuberculosis and Y. enterocolitica were consistent with the pathogenic profile characteristic of these two Yersinia species. No significant epidemiological correlation was found in the grouping of Y. bercovieri, Y. rohdei, Y. intermedia Y. mollaretii and Y. ruckeri according to HRMA results. Moreover, the HRMA-based method develop here was able to separate the Y. pseudotuberculosis and Y. enterocolitica species. The HRMA assay developed in this study can be used as an alternative for the genotyping and the differentiation of Y. pseudotuberculosis and Y. enterocolitica. This method can also complement sequence-based methods and facilitate epidemiological studies of these two Yersinia species.
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Darbandy, Ashna. "Optimization of High Resolution Melting Analysis for Detection of KRAS Gene Mutations". Thesis, Uppsala University, Department of Medical Biochemistry and Microbiology, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-130751.
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Background: Mutations of the KRAS oncogene occur in a variety types of human tumors. By assessing the mutation status of KRAS, clinicians can predict patient response to anti-EGFR therapy such as cetuximab (Erbitux®) or panitumumab (Vectibix®) in patients with metastatic colorectal cancer. The aim of this study was to optimize a real-time PCR method followed by high resolution melting analysis (HRM) in a single step for detection of most common mutations within the KRAS gene. Methods: Seven DNA samples with predefined KRAS mutations and 19 tumor samples from patients with metastatic colorectal cancer were used. KRAS mutation detection was performed by direct sequencing as well as HRM. Optimization was performed using touchdown PCR and co-amplification at lower denaturation-temperature PCR. Results: All DNA samples were successfully analyzed with direct sequencing and HRM. Moreover, the improved amplification efficiency and sensitivity was achieved using optimized PCR run protocol. Conclusion: HRM is a simple, inexpensive and reliable method for mutation detection within KRAS. By applying HRM as prescreening method would help reduce labour, time and costs.
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Burrows, Adria Michelle. "A comparative ancestry analysis of Y-chromosome DNA haplogroups using high resolution melting". University of the Western Cape, 2018. http://hdl.handle.net/11394/6536.
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>Magister Scientiae - MScThe objective of this study is to deduce paternal ancestry using ancestry informative single nucleotide polymorphisms (SNPs) by means of High Resolution Melting (HRM). This was completed by producing a multiplex system that was designed in a hierarchical manner according to the YSNP tree. This project mainly focused on African ancestry and was used to infer paternal ancestral lineages on the Johannesburg Coloured population. South Africa has a diverse population that has ancestral history from across the globe. The South African Coloured population is the most admixed population as it is derived from at least five different population groups: these being Khoisan, Bantu, Europeans, Indians and Southeast Asians. There have been studies done on the Western Cape/ Cape Town Coloured populations before but this study focused on the Johannesburg Coloured population.
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Michelle, Burrows Adria. "A comparative ancestry analysis of Y-chromosome DNA haplogroups using high resolution melting". University of the Western Cape, 2018. http://hdl.handle.net/11394/6489.
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Magister Scientiae - MSc (Biotechnology)The objective of this study is to deduce paternal ancestry using ancestry informative single nucleotide polymorphisms (SNPs) by means of High Resolution Melting (HRM). This was completed by producing a multiplex system that was designed in a hierarchical manner according to the YSNP tree. This project mainly focused on African ancestry and was used to infer paternal ancestral lineages on the Johannesburg Coloured population. South Africa has a diverse population that has ancestral history from across the globe. The South African Coloured population is the most admixed population as it is derived from at least five different population groups: these being Khoisan, Bantu, Europeans, Indians and Southeast Asians. There have been studies done on the Western Cape/ Cape Town Coloured populations before but this study focused on the Johannesburg Coloured population. The first step was to design the multiplex system. This was done by using inhouse SNPs. A total of seven multiplexes were designed and optimised, each consisting of two, three or four different SNPs respectively. A total of 143 saliva and buccal samples were collected from male Johannesburg Coloureds. DNA was extracted from the saliva samples using an optimised organic method. DNA was extracted from the buccal samples using an optimised salting out method. DNA was successfully extracted from 77 of the male samples. A total of 69 samples were screened using Multiplex 1; of the 69 samples 56 samples were successfully screened to infer the paternal lineage of the samples. The results show that the most frequent haplogroup of the Johannesburg male samples was haplogroup CF (39%). The second most frequent haplogroup was haplogroup DE (38%). Under further analysis of haplogroup DE it was seen that 37% of those samples were derived for the haplogroup E1b1b.
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PIMENTEL, ROMERO CESAR HUGO. ""INNOVATIVE SIGNAL PROCESSING TECHNIQUES IN BIOENGINEERING: COMPRESSED SENSING AND HIGH RESOLUTION DNA MELTING ANALYSIS"". Doctoral thesis, Università degli studi di Ferrara, 2021. http://hdl.handle.net/11392/2487913.
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The first part starts with an introduction of the Compressed Sensing (CS) theory. After that, an overview of the state-of-the art of some CS adaptations by using additional priors in the different stages is presented in order to explain the new CS adaptation proposed in this work. Some of the CS adaptations reviewed are confronted using synthetic signals, synthetic electrocardiograph (ECG) signals and electroencephalographic (EEG) signals. The second part provides general concepts of biology and current developments in bioinformatics to read and and analyze the DNA. Questions like what a sequencer does?, What kind of data it produces? and How can be analyzed this data? are intended to be answered. Another reliable technique used in the analysis of the DNA without sequencing is the High Resolution Melting (HRM) curves analysis, this technique is used to find differences between two strands of DNA. HRM is also discussed to finally design a HRM analysis software.La prima parte inizia con un'introduzione alla teoria del Compressed Sensing (CS). Successivamente, viene presentata una panoramica dello stato dell'arte di alcuni adattamenti CS utilizzando nelle diverse fasi, questo per spiegare il nuovo adattamento CS proposto in questo lavoro. Alcuni degli adattamenti CS esaminati vengono confrontati utilizzando segnali sintetici, segnali di elettrocardiografo sintetico (ECG) e segnali elettroencefalografici (EEG). La seconda parte fornisce concetti generali di biologia e sviluppi attuali della bioinformatica per leggere e analizzare il DNA. Domande come Cosa fa un sequencer? Che tipo di dati produce? e Come possono essere analizzati questi dati? sono destinati a ricevere una risposta. Un'altra tecnica affidabile utilizzata nell'analisi del DNA senza sequenziamento è l'analisi High Resolution Melting (HRM) curves, questa tecnica viene utilizzata per trovare differenze tra due filamenti di DNA. Si studia anche le HRM curves per progettare finalmente un software di analisi.
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Tsang, Ho-yin y 曾皓言. "Detection of clinically silent beta-globin gene mutations in Chinese using high resolution melting analysis". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hub.hku.hk/bib/B48334182.
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Mutations in the beta-globin (β-globin) gene cause beta-thalassaemia (β-thalassaemia).The screening strategy for β-thalassaemiais based on the value of mean corpuscular volume (MCV) from the complete blood count (CBC) data. Current laboratory practice considers blood samples with MCV higher than 80fL as normal. No further assessment will be done on these samples. However, there are clinically silent β-globin gene mutations with MCV higher than 80fL, for example, heterozygous haemoglobin E (HbE). The importance of finding out this kind of mutations is due to the serious outcome when they occur together with classic β thalassaemia mutations in compound heterozygous states, which may produce a condition mimicking β thalassaemia major.
The method used to recognize the presence of clinically silent β-globin gene mutations should be robust and with high sensitivity. High resolution melting (HRM) is a suitable technique to screen gene mutations. It is fast and convenient. The process is completed in a closed system without any post PCR manipulation. The sensitivity is up to a single nucleotide change. Using HRM for mutations screening followed by confirmation with sequencing can reduce time and cost of testing clinically silent β-globin gene mutations on a large scale.
This study first shows the ability of HRM in detecting various types of β-globin gene mutations. The technique is then applied to detect clinically silent β-globin gene mutations in a group of high school students with normal CBC data. Mutations with different clinically significance were found. The frequency of mutation found in the samples of the study suggests that screening for β-globin gene mutation may be worthwhile in subjects with MCV higher than 80fL.published_or_final_version
Pathology
Master
Master of Medical Sciences
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Ho, Sophia KW y 何廣慧. "Detection of clinically silent alpha-globin gene mutations in Chinese using high resolution melting analysis". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2014. http://hdl.handle.net/10722/206558.
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α-thalassemia is an inherited globin gene disorder commonly found among the Chinese population. It is composed of both non-deletional and deletional α-globin gene mutations. Classical α-thalassemia presents with red cell microcytosis but silent cases with a normal mean corpuscular volume (MCV) are also seen. Routine laboratory testing methods for large-scale detection of silent α-thalassemia mutations are onerous and time-consuming. Furthermore, methods such as denaturing high performance liquid chromatography (HPLC) or denaturing gradient gel electrophoresis (DGGE) for scanning of point mutations are costly and they require post-PCR separation. High resolution melting (HRM) analysis is an economical, sensitive, and fast method for large scale point mutation scanning. Contamination is significantly reduced with HRM because the process is performed in a closed-tube environment and does not require post-PCR manipulation. We used HRM and multiplex gap-PCR analysis to determine the prevalence of silent α-thalassemia carriers in Hong Kong.
Of the 223 hematologically normal blood samples scanned by Roche LightCycler 480®, HRM did not show any sample with a non-deletional α-globin gene mutation of clinical significance. α-multiplex gap-PCR analysis revealed 36 samples (16.1%) with single α-globin gene deletions. The detection of single α-globin gene deletions in samples with a MCV greater than 80 fL indicates that the previously reported prevalence of α-thalassemia mutations in our Chinese population based on MCV screening is under-estimated. The data also suggest that non-deletional α-thalassemia mutations presenting with a normal MCV are very rare, and they most likely present with microcytosis.
The fact that most silent α-thalassemia mutations are due to large deletions supports the use of traditional molecular techniques such as gap-PCR for their detection. HRM can be used as an adjunct tool for large-scale population screening of non-deletional mutations. This study provides more accurate data on the prevalence of silent α-thalassemia carriers in the Hong Kong Chinese population. The information will facilitate genetic counseling and risk assessment in families carrying α-thalassemia mutations.published_or_final_version
Pathology
Master
Master of Medical Sciences
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Ozbak, Hani. "The application of High Resolution Melting Analysis (HRMA) for rapid detection of bacteria responsible for bloodstream infections". Thesis, University of Manchester, 2013. https://www.research.manchester.ac.uk/portal/en/theses/the-application-of-high-resolution-melting-analysis-hrma-for-rapid-detection-of-bacteria-responsible-for-bloodstream-infections(b3d5c15b-9541-44c2-873c-f7a32fc60282).html.
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Background: The diagnosis of bloodstream infection is a significant challenge for healthcare providers and is often associated with severe illness (sepsis) and poor outcomes. Rapid detection and identification of pathogens followed by characterisation of antibiotic resistance could help direct early treatment and improve patient care. Standard blood culture methods, which usually take 2-5 days to complete, can confirm if there is a bacteraemia or not in suspected patients. However, molecular approaches have been developed and are being increasingly investigated to overcome disadvantages of culture. One of the main potentials of molecular techniques is that they should be able to identify pathogens within a short time which could help clinicians treat patients earlier with rational antimicrobial therapy and limit overuse of antibiotic exposure. Objectives: To present the development and optimisation of a simple, rapid and cost-effective Real Time PCR methods combined with a High Resolution Melting Analysis (HRMA) approach, to detect and identify common bacteria associated with bloodstream infections. Approach: 16S rRNA and Gram classification primers were used on a broad range real-time PCR for molecular Gram typing and HRMA in a single run. Differentiation of bacterial species was achieved using a multi-parameter, decision-tree approach based on Gram typing, grouping according to melting temperature (Tm) and sequential comparisons of melting profiles (Curve shapes) against reference organisms. Findings: A preliminary validation was undertaken by blinded analysis of 53 consecutive bloodstream isolates from a clinical microbiology laboratory. 50 isolates contained organisms present on the panel and 96% of these were identified correctly at genus or species level. A correct Gram classification was reported for all 53 isolates. The strategy of amplification of the bacterial signal to an appropriate level using a short term pre-culture system (STPCS) for up to 12 hours prior to HRMA analysis significantly improved the overall sensitivity of the assay in spiked blood. Conclusion: This study suggests that a PCR-HRMA approach could be used as an alternative cheap approach to other molecular approaches for rapid detection and identification of bacteria responsible for >95% of bloodstream infections especially when combined with a Short Term Pre-Culture System (STPCS). Such development together with the current standard culture-based methods could allow clinicians to establish more effective management and treatment of patients with suspected bloodstream infection at an earlier stage than is possible with only current culture-based approaches.
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Loiacono, M. "MOLECULAR CHARACTERIZATION BY RT-REAL TIME PCR AND HIGH RESOLUTION MELTING ANALYSIS FOR FOOD SAFETY AND VETERINARY DIAGNOSTICS". Doctoral thesis, Università degli Studi di Milano, 2015. http://hdl.handle.net/2434/338720.
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This PhD thesis is the outcome of a range of activities and experimental results aimed to a better characterization of the risk that Escherichia coli and other microrganisms and parasites may pone to the health of animals and finally humans. One of the main activity of the present project was based on the test hypothesis that the virulence profile of E. coli strains toward bovine mammary gland can be modulated by the interaction with the host cells. These hypothesis were tested through a gene expression study of some virulence factors of six E. coli strains when co-cultured with a bovine mammary cell line, since the in vitro models represent both an essential tool to investigate the biological mechanistic of mastitis, and an efficient alternative to animal experiments. Preliminarly, a meta-analysis of existing literature studies on the available bovine mammary cell lines was performed, resulting in the selection of MAC-T as the most responsive cell line to bacteria causing mastitis. The E. coli strains used for the coculture experiments with MAC-T cells were isolated from different types of bovine mastitis (acute, chronic and undetermined) and from a VTEC food-borne strain associated to human clinical disease (O157). An upregulation of the virulence factor eae (intimin) in all but one the analyzed mastitis strains following co-culture with MAC-T cell line was detected through RT-reat time PCR, and also the adherence virulence factor ycd and the b12 gene were upregulated in some strains, overall suggesting the possibility that mastitic E. coli strains can acquire a more risky molecular profile when exposed to the bovine mammary cells. This finding may have clear implications on the risk assessment related to the E. coli strains in bovine mammary tissue and milk. In addition, with the aim to improve the current methodologies for foodborne risk analysis linked to E. coli, the project activity provided a preliminary research for the setup and validation of new protocols based on real time PCR-High Resolution Melting Analysis, a widely used technique to target sequence polymorphisms of the same gene in different species without the need to perform DNA sequencing or to use species-specific probes, to help the identification of putative verocytotoxic status in E. coli strains of O26 serogroup, and other serotypes, isolated from bovine milk.
Since the applications of HRMA for the characterization of microorganisms can not be limited to food safety, but can be developed for a large number of issues linked to general veterinary diagnostics, among the objectives of this PhD project some new real-time PCR-HRMA coupled methods were also developed, providing a contribution to the advancement of the existing molecular tools for sensitive and effective species identification, or variant/mutation screening, applied to different foodborne and veterinary pathogens. Thus, new HRMA-based protocols were designed and tested for the identification of Pseudomonas spp responsible for chromatic alterations in mozzarella cheese, for the detection and differentiation of Dirofilaria repens and D. immitis in canine blood samples, for the detection of the mutation site associated to FQ resistance in Staphylococcus pseudintermedius isolated from canine diagnostic samples, and for discrimination of the two most common microsporidial parasites in honeybees, Nosema apis and N. ceranae. Overall, these new HRMA-based assays could represent additional tools for epidemiological studies, routine disease assessment and therapeutical decisions.
The possibility to identify the presence of risk-predictive SNPs in E. coli isolates using these newly established HRMA-based protocols is a novel, and simpler, opportunity with respect to the current, and more complex, surveillance strategies that are based on the amplification of stx genes together with other virulence factors for the evaluation of VTEC status. In the future, a possible way forward of this research is represented, on one side, by the deeper assessment of the reciprocal modulation between E. coli mastitis-derived strains and immortalized MAC-T cells using high-throughput RNA sequencing, and on the other side by a large scale validation of the HRMA-based evaluation of risk-predictive SNPs in order to improve the current approaches. And overall, the established HRMA-based protocols when extensively validated would be highly suitable for routine veterinary diagnostics applied to field investigation, as quick and sensitive single step protocols allowing specific and sensitive detection of the targets with shorter analysis time and reduced cost, in parallel or in alternative to the classical approaches.
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Raphela, Mashikoane Pinky Jane. "Molecular characterization of Mycoplasma synoviae in chickens in South Africa using single-stranded conformation polymorphism and high-resolution melting curve analysis of the vlhA gene". Diss., University of Pretoria, 2012. http://hdl.handle.net/2263/26196.
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Mycoplasma synoviae (Ms) causes respiratory infection and synovitis in chickens and turkeys and is an economically important pathogen of poultry worldwide. It is critically important to characterize Ms strains, especially in countries in which poultry flocks are vaccinated with the live attenuated Ms strain MS-H. Vaccination with this vaccine may cause sero-conversion and persistence of the vaccine strain in the respiratory tract and will frequently result in positive Ms cultures and PCR results. Vaccination of flocks therefore complicates the diagnosis of Ms by the presence of detectable antibodies in the blood. Many diagnostic techniques cannot distinguish between the vaccine strain and wild type strain. Single stranded conformation polymorphism (SSCP) and real-time PCR with high melting curve (HRM) analysis can discriminate between the different Ms strains obtained from the field and also distinguish them from the live vaccinestrains. These techniques provide effective tools for the further study of the epidemiology and spread of Ms strains in chickens in South Africa. This project was undertaken to establish whether SSCP and HRM analyses can be used effectively to discriminate between Ms field isolates and the vaccine strain. Mycoplasma synoviae DNA was extracted from samples and conventional PCR was performed using oligonucleotide primers complementary to the single-copy conserved 5’ end of the variable lipoprotein and haemagglutinin encoding gene (vlhA). Twenty six samples were separated by agarose gel electrophoresis and prepared for SSCP and real-time PCR and HRM curve analysis. Results obtained from SSCP were compared to real-time PCR/HRM. Differences obtained by SSCP and melting curve analysis between different isolates were confirmed by sequencing. Results obtained from the different techniques differentiated the strains from the vaccine strain (isolate Ms10), which had a different melting temperature to the others and a different band pattern on the SSCP gel. These results confirmed that HRM and SSCP methods can be used to detect and discriminate between Mycoplasma synoviae field isolates and the vaccine strain.Dissertation (MSc)--University of Pretoria, 2012.
Veterinary Tropical Diseases
unrestricted
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Brecht, Gadean. "Genetic analysis of mitochondrial DNA within Southern African populations". UWC, 2020. http://hdl.handle.net/11394/7365.
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>Magister Scientiae - MScAs human beings we are curious about our origin and ancestry. A curiosity has led to an investigation of human evolution and expansion across the world by means of population genetics and phylo-genetics by evaluating a region in Southern Africa that is largely unknown. The objective of this study was to develop a quick, inexpensive and accurate hierarchical diagnostic screening system of the MtDNA phylogenetic tree, AI-SNPs in the mtDNA genome by using High Resolution Melting analysis to evaluate the population composition and ancestral haplogroups of Southern African populations in Limpopo. The admixture between the ‘Khoesan’ hunter-gatherers, herders and the Bantu speaking populations led to population growth and expansion in Limpopo. This has contributed to populations settling in Limpopo and has thus shaped the ancestral contemporary populations. No research on these individuals residing in Limpopo has been done before, thus an investigation of their ancestral origin was necessary. A total of 760 saliva samples were collected from individuals residing in Limpopo. Only 500 saliva samples were extracted by means of an optimized salting out technique. Five hundred extracted genomic samples were genotyped by means of a quick, inexpensive High-resolution melting analysis. Of the 500 samples, the genotyping results showed 95 individuals derived for the L3 haplogroup which gives a 19% ratio of individuals screened with Multiplex 1. Only 56 individuals were derived for the L1 haplogroup, which gives a percentage of 11%. A total of 249 individuals were derived for the L0 haplogroup, making up a 50% of the total individuals genotyped. Only 100 samples were derived for L0a, making up 20% of individuals screened with Multiplex 1. Of the 95 samples derived for the L3 haplogroup, the results showed 87 individuals to be ancestral for both M and N, making up 91.57% of individuals screened with Multiplex 2. http://etd.uwc.ac.za/. In population genetics using SNPs to infer population history and ancestral origin has become significant, this study allowed researchers to evaluate population groups by investigating their genetic markers and the application of the results allowed for downstream analyses. Finally, this study provides a quick and simple screening method for the selection of lineages that are of interest for further studies.
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Al_griw, Huda Hm. "Molecular detection of bloodstream pathogens in critical illness". Thesis, University of Manchester, 2012. https://www.research.manchester.ac.uk/portal/en/theses/molecular-detection-of-bloodstream-pathogens-in-critical-illness(5f143a31-3694-454c-8940-5ae434f1eb31).html.
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Background: Critically ill patients are at particular risk of developing bloodstream infection. Such infections are associated with the development of sepsis, leading to a marked increase in mortality rate. Early detection of the causative organism and appropriate antibiotic treatment are therefore critical for optimum outcome of patients with nosocomial infection. Current infection diagnosis is based on standard blood culture techniques. However, microbiological culture has a number of limitations, not least that it takes several days to confirm infection and is therefore not useful in directing the early treatment with antibiotics. New techniques based on the detection of pathogen DNA using real-time polymerase chain reaction (PCR) technology have the potential to address these limitations but their clinical utility is still to be proved. Objectives: Develop and evaluate novel PCR-based approaches to bloodstream infection diagnosis in critical illness based on detection and identification of bacterial and fungal DNA in blood. Methods: A range of commercial and 'in-house' PCR-based assays for detection of bacterial and fungal DNA were developed and/or optimised for use in clinical blood samples. These included LightCycler SeptiFast, a CE-marked multi-pathogen assay for common bloodstream pathogens, BactScreen and GramScreen, broad spectrum bacterial assays based on 16S rRNA gene and real-time PCR assays developed to detect a range of clinically important fungal pathogens. Novel approaches to speciation of pathogen DNA using melting temperature (Tm) profiling and high resolution melting analysis (HRMA) were developed. Clinical evaluation of assays was either on blinded clinical isolates or blood samples from critically ill patients with clinical suspicion of bloodstream infection against conventional microbiological culture. Several techniques aimed at improving extraction of pathogen DNA from blood were also investigated. Results: The CE-marked commercial assay SeptiFast showed analytical sensitivity and specificity of 79% and 83% respectively. Concordance with positive culture results was good but high levels of 'false positives' were detected possibly attributed to detection of free pathogen DNA not associated with viable pathogens. The predictive value of a negative SeptiFast test was 98% suggesting that absence of pathogen DNA is a strong indicator of absence of infection. Further studies were aimed at detailed optimisation and validation of 16S rRNA gene real-time PCR assays for bacterial DNA. BactScreen and GramScreen were able to detect a broad range of clinically important bacteria down to <50 CFU/ml blood. A preliminary comparative evaluation against SeptiFast showed BactScreen gave excellent concordance with blood culture results with minimal false positive results compared to SeptiFast. Efficient extraction of pathogen DNA was shown to be a key factor in determining analytical sensitivity and several protocols were evaluated. Low cost approaches to speciation of bacterial DNA were developed by combining broad range real-time PCR with HRMA. A novel HRMA method based on Tm profiling was shown to identify 89% and 96% of blinded clinical isolates at species or genus level respectively. Real-time PCR/HRMA approaches were also successfully developed for detection and identification of fungal pathogens including a range of Candida and Aspergillus species associated with bloodstream fungal infection. Conclusions: These studies have highlighted some of the key factors that need to be considered when developing and validating PCR based assays for pathogen DNA detection in blood. A set of novel tools have been developed for rapid detection and identification of bacterial and fungal pathogens that could address the challenges of infection diagnosis based on pathogen DNA detection. Further work is required, not least in development of more efficient pathogen DNA extraction and detailed clinical validation but the tools described here have the potential to provide cost effective solutions to aid infection diagnosis that would be complementary to current culture-based methods. The provision of time critical information could have a positive impact on clinical decision-making leading to more effective management and treatment of patients with suspected bloodstream infection.
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Sedláková, Lucie. "Analýza DNA izolované z různých typů probiotických výrobků s využitím PCR v reálném čase a HRM analýzy". Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2016. http://www.nusl.cz/ntk/nusl-240562.
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The aim of this diploma thesis was to introduce real-time PCR with high-resolution melting analysis for Bifidobacterium species. Currently a small number of publication, dealing with identification of Bifidobacterium species using high-resolution melting analysis, is available. According to publications dealing with identification of lactic acid bacteria were selected primers P1V1 and P2V1, LAC1 and LAC2, LsppUPF and LsppUPR, V3F and V3R, V6F and V6R. Using this primers bacterial DNA was amplified by real-time PCR with high-resolution melting analysis. After evaluation of the measured results efficiency of selected primers was verified on DNA izolated from complex sample of probiotic product. After further optimisation real-time PCR with high-resolution melting analysis could be suitable using selected primers for Bifidobacterium species.
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Pires, Elisabete Sofia Videira. "Real-Time PCR, High Resolution Melting - aplicações forenses". Master's thesis, Universidade de Aveiro, 2012. http://hdl.handle.net/10773/10747.
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Mestrado em Biologia Molecular e CelularApontada como a maior revolução científica na área forense desde a descoberta das impressões digitais, a identificação humana por meio da análise do DNA tornou-se uma poderosa ferramenta de investigação, auxiliando na elucidação de casos forenses, baseando-se cientificamente na existência de polimorfismos genéticos ao longo do genoma em indivíduos diferentes, que faz com que cada pessoa possua um código genético único. Com a introdução da real-time PCR nas investigações forenses, tornou-se possível uma análise sensível e específica de regiões polimórficas tanto no genoma nuclear como no mitocondrial, a partir de quantidades ínfimas de DNA obtidas de amostras altamente degradadas ou com baixo número de cópias. A quantificação do DNA é um procedimento importante na análise forense e deve ser efetuado, previamente, a qualquer análise de DNA. A união entre a bioinformática e a genética forense propiciou a criação de métodos de análise específicos, como a HRM, muito útil na genotipagem de SNPs, de extrema importância na investigação forense. Foi elaborada uma revisão bibliográfica com o objetivo de conhecer as aplicações forenses da real-time PCR e os respetivos métodos, tendo se confirmado então a aplicabilidade deste método na área forense.
Listed as the greatest revolution in forensic science since the discovery of fingerprints, identification by analyzing human DNA has become a powerful research tool, helping to elucidate forensic cases, scientifically based on the existence of genetic polymorphisms throughout the genome at different individuals, which causes that each person has a unique genetic code. With the introduction of real-time PCR in forensic investigations, it became possible a sensitive and specific analysis of polymorphic regions both in the mitochondrial and nuclear genome, from minute quantities of DNA obtained from samples highly degraded or low copy number. The quantification of DNA is an important procedure in forensic analysis and must be made in advance to any DNA analysis. The union between forensic genetics and bioinformatics led to the creation of specific analysis methods, such as HRM, very useful in scanning and genotyping of SNPs, of utmost importance in forensic investigation. A literature review has been prepared in order to meet the forensic applications of real-time PCR and related methods, and so been confirmed the applicability of this method in the forensic field.
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Hamano, Yuya. "Forensic age prediction by use of methylation-sensitive high resolution melting". Kyoto University, 2018. http://hdl.handle.net/2433/232076.
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Konečná, Jana. "PROBIOTICKÉ GENY POTRAVINÁŘSKY VÝZNAMNÝCH BAKTERIÍ MLÉČNÉHO KVAŠENÍ". Doctoral thesis, Vysoké učení technické v Brně. Fakulta chemická, 2019. http://www.nusl.cz/ntk/nusl-402108.
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Isolation of deoxyribonucleic acid (DNA) is an important step in the molecular diagnostics of microorganisms. A high quality of isolated DNA is necessary for DNA amplification by the polymerase chain reaction (PCR). The conventional DNA isolation using phenol chloroform extraction and DNA precipitation in ethanol is time consuming and requires the use of toxic phenol. Magnetic separation techniques using magnetic solid particles are one of modern methods to speed up the nucleic acids isolation. The aim of this work was to use two different types of magnetic particles for solidphase DNA extraction. The amounts of DNA in separation mixtures were measured using ultraviolet spectrophotometry (UV). The first experimental conditions were tested on chicken erythrocytes DNA. Phosphate buffer (pH 7, 7.6 and 8) was used for adsorption of DNA on magnetic particles. It was shown that approximately almost one half of DNA was adsorbed to the particles. The elution conditions of DNA were also optimized. Secondly, bacterial DNA was tested. This DNA eluted from the particles was in PCR ready quality. High resolution melting (HRM) curve analysis is a simple, low-cost method for amplicon discrimination and easy connection with real-time polymerase chain reaction (PCR). In this contribution, we report rapid species identification of strains belonging to the Lactobacillus group using HRM-PCR. Three different DNA isolation methods were used in this work: phenol extraction, separation using magnetic particles and commercial kit. Ten sets of targeted gene fragments primers (LAC1 – LAC2, LAC2 – LAC4, P1V1 – P2V1, Gro F – Gro R, 3BA-338f – Primer 1, V1F – V1R, CHAU - V3F – CHAU - V3R, CHAU - V6F – CHAU - V6R, poxcDNAFw – poxPromRVC, poxcDNAFw – poxPromRVT) were tested for amplification of the 16S rRNA gene. Use of GroF/R and LAC2/4 primers pairs successfully identify strains belong to the Lactobacillus group. The variance between used extraction methods for evidence of HRM curves was found.
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Konečná, Jana. "Využití molekulárně biologických technik pro identifikaci a analýzu probiotických bakterií". Doctoral thesis, Vysoké učení technické v Brně. Fakulta chemická, 2019. http://www.nusl.cz/ntk/nusl-402114.
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Isolation of deoxyribonucleic acid (DNA) is an important step in the molecular diagnostics of microorganisms. A high quality of isolated DNA is necessary for DNA amplification by the polymerase chain reaction (PCR). The conventional DNA isolation using phenol-chloroform extraction and DNA precipitation in ethanol is time-consuming and requires the use of toxic phenol. Alternative method of DNA isolation is use of commercially available kits which, however, are expensive and their efficiency is low. Magnetic separation techniques using magnetic solid particles are one of modern methods to speed up the nucleic acids isolation. The aim of this work was to use two different types of magnetic particles for solid-phase DNA extraction. Magnetic microparticles P(HEMA – co – GMA) containing –NH2 group and nanoparticles PLL, whitch contains polylysine. The amounts of DNA in separation mixtures were measured using ultraviolet spectrophotometry (UV). The first experimental conditions were tested on chicken erythrocytes DNA. Phosphate buffer (pH 7, 7.6 and 8) was used for adsorption of DNA on magnetic particles. It was shown that approximately almost one half of DNA was adsorbed on the particles. The elution conditions of DNA were also optimized. Secondly, bacterial DNA was tested. After optimalization, the developed method was used for DNA isolation from real food supplements. This DNA eluted from the particles was in PCR ready quality. High resolution melting (HRM) curve analysis is a simple, low-cost method for amplicon discrimination and easy connection with real-time polymerase chain reaction (PCR). In this thesis, we report rapid species identification of strains belonging to the Lactobacillus group using HRM-PCR. Three different DNA isolation methods were used in this work: phenol extraction, separation using magnetic particles and commercial kit. Ten sets of targeted gene fragments primers (LAC1 – LAC2, LAC2 – LAC4, P1V1 – P2V1, Gro F – Gro R, 3BA-338f – Primer 1, V1F – V1R, CHAU - V3F – CHAU - V3R, CHAU - V6F – CHAU - V6R, poxcDNAFw – poxPromRVC, poxcDNAFw – poxPromRVT) were tested for amplification of the 16S rRNA gene. Use of GroF/R and LAC2/4 primers pairs successfully identify strains belong to the Lactobacillus group. The variance between used extraction methods for evidence of HRM curves was found.
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Salgado, Isa Sofia Ribeiro. "Deteção de mutações nos genes JAK2 e MPL por high resolution melting". Master's thesis, Universidade de Aveiro, 2013. http://hdl.handle.net/10773/12628.
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Mestrado em Biologia AplicadaAs NMPs representam um grupo heterogéneo de doenças clonais caracterizadas pela expansão e produção excessiva de eritrócitos, granulócitos e/ou plaquetas sanguíneas na medula óssea. Em 2005, foi identificada a mutação V617F no gene JAK2 numa elevada frequência de doentes com NMP, em especial nos doentes com PV (65-97%), TE (23-57%) e MFP (35-57%). A deteção da mutação V617F no gene JAK2 e de outras mutações funcionalmente similares, isto é, mutações no exão 12 do gene JAK2 e no exão 10 do gene MPL, foram recentemente incluídas pela Organização Mundial de Saúde, nos critérios de diagnóstico para a PV, TE e MFP. Várias técnicas têm sido descritas e aplicadas à pesquisa destas mutações. A técnica de AS-PCR (PCR alelo-específico) é considerada uma técnica de diagnóstico capaz de detetar uma mutação heterozigótica presente em apenas 1-3% das células. Recentemente, o HRM foi descrito como uma técnica simples, rápida, de baixo custo e com elevada sensibilidade e especificidade na identificação e/ou deteção de mutações. Este estudo teve como principal objetivo avaliar a eficácia da técnica de HRM na deteção da mutação V617F-JAK2, das mutações no exão 12 do gene JAK2 e do exão 10 do gene MPL, numa série de 160 amostras de doentes com diagnósticos de NMP. A técnica de HRM demonstrou uma especificidade de 100% e uma sensibilidade ligeiramente inferior (98,4%) na deteção da mutação V617F, quando comparada com a técnica utilizada por rotina no GDPN para a deteção desta mutação (AS-PCR). Na pesquisa de mutações no exão 12 do gene JAK2 e exão 10 do gene MPL, a técnica de HRM permitiu detetar 100% dos casos com mutação. Os resultados deste estudo sugerem que o HRM tem uma utilização limitada na deteção da mutação V617F do gene JAK2, embora se tenha revelado uma técnica adequada ao rastreio rápido das mutações do exão 12 do gene JAK2 e do exão 10 do gene MPL. No presente estudo, foram detetadas mutações nos genes JAK2 e MPL em 80,6% dos doentes com PV, em 32,0% dos doentes com TE, em 33,3% dos doentes com MFP e em 33,3% dos doentes com NMP não classificadas.
Myeloproliferative neoplasms (MPNs) are a heterogeneous group of clonal diseases characterized by increased and excessive proliferation of erythrocytes, granulocytes and/or platelets in the bone marrow. In 2005, several groups identified the presence of the V617F mutation in the JAK2 gene in a significant proportion patients with PV (65-97%), ET (23-57%) and PMF (35-57%). Detection of the JAK2 mutation or other functionally similar mutation, such as JAK2 exon 12 mutations or MPL exon 10 mutations, have recently been included in the essential diagnostic criteria for PV, ET and PMF by the World Health Organization. Several techniques have been used to detect these mutations. AS-PCR (allele specific PCR) is considered a diagnostic tool capable of detecting a heterozygous mutation present in only 1-3% of mutated cells. Recently, HRM was described as a simple, fast and cost effective technique with high sensitivity and specificity that allows the detection and identification of mutations. The present study aimed at the evaluation of HRM as a diagnostic tool to detect JAK2-V617F, JAK2 exon 12 mutations or MPL exon 10 mutations, in 160 samples of MPNs patients. HRM revealed a 100% specificity and a slightly lower sensitivity (98,4%) in the V617F mutation detection when compared to AS-PCR. HRM detected all positive cases with JAK2 exon 12 mutations or MPL exon 10 mutations. Our results suggest that HRM is of limited use to detect the JAK2-V617F mutation. However, it is a suitable technique for mutation screening of JAK2 exon 12 mutations or MPL exon 10 mutations. In this study, JAK2 and MPL mutational frequency was 80,6% in PV, 32,0% in TE, 33,3% in PMF and in unclassifiable MPNs patients.
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ALMEIDA, F. A. N. "AUTÊNTICAÇÃO de Ginseng Brasileiro Utilizando a Técnica de High Resolution Melting (hrm)". Universidade Federal do Espírito Santo, 2018. http://repositorio.ufes.br/handle/10/7113.
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Previous issue date: 2018-02-20À medida que a indústria de plantas medicinais cresce, a autenticidade de seus produtos é uma questão de segurança do consumidor e não pode ser negligenciada. O Ginseng brasileiro, por exemplo, se refere a duas espécies distintas, Pfaffia glomerata e Hebanthe eriantha. Apesar da similaridade entre essas duas espécies, o que dificulta a identificação morfológica, elas possuem propriedades químicas e farmacológicas distintas. Foi proposto que a técnica de High Resolution Melting (HRM) como uma ferramenta para discriminar e identificar as espécies P. glomerata e H. eriantha utilizando uma região do gene matK e rbcL. Para tal, foram adquiridas seis amostras referência, três de cada uma das espécies citadas, e 60 amostras comerciais, vendidas como Ginseng brasileiro, por meio de compra física ou online. O DNA foi extraído pelos métodos CTAB e por Kit. Os primers desenhados foram testados através da amplificação por PCR convencional e confirmado em gel de poliacrilamida antes da padronização da amplificação por PCR-HRM e por fim os resultados foram comparados aos de sequenciamento. Foram desenvolvidos três primers dois para o gene matK, HRM-matKD e HRM-matK, e um para o gene rbcL, HRM-rbcL. Durante a padronização notou-se a influência da temperatura de anelamento e concentração do primer no resultado da corrida, entretanto o método de extração não alterou os resultados. A análise de HRM mostrou que o primer HRM-matKD foi o que atendeu ao objetivo proposto apresentando alta sensibilidade (92-93%) e especificidade (100%) comparado ao método de sequenciamento. Foi possível validar a técnica de HRM utilizando amostras comerciais previamente sequenciadas, o que nos permite afirmar que a técnica de HRM pode ser utilizada para discriminar H. eriantha e P. glomerata. HRM é um método rápido e de baixo custo, sendo uma ferramenta confiável para identificação de espécies de Ginseng brasileiro.
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Knápková, Monika. "Použití vysokorozlišovací analýzy křivek tání ke studiu baktérií mléčného kvašení". Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2019. http://www.nusl.cz/ntk/nusl-401890.
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Currently, there is a growing interest in the use of probiotic products, and there are many of them in the market. With the growing interest, greater emphasis is placed on the identification of declared probiotic microorganisms. Precise identification of microbial composition is often a difficult task and it requires more advanced methods especially in the field of molecular diagnostics. The diploma thesis was focused on the verification of the presence od declared probiotic microorganisms in probiotic food supplements GS Laktobacily Forte 21, Biopron 9 Premium and Linex® Forte. DNA was isolated from the complex matrices by phenol extraction, commercial kit and magnetic carriers F79/L3-PLL in the quality suitable for PCR. Subsequently, the isolated DNA was amplified by real-time polymerase chain reaction using genus- and species-specific primers. The specific PCR product was subjected to agarose gel electrophoresis, whereas species identification was not always in compliance with the data declared by producers. The next part of the thesis was focused on polymerase chain reaction with high-resolution melting analysis to distinguish bacterial strains belonging to the Lactobacillus group and to identify probiotic microorganisms present in the complex matrices of the probiotic food supplements. Eight primer sets were tested (V1F HRM a V1R-HRM, CHAU-V3F a CHAU-V3R, CHAU-V6F a CHAU-V6R, LAC2 a LAC4, LAC1 a LAC2, P1V1 a P2V1, poxcDNAFw a poxPromRVC, poxcDNAFw a poxPromRVT). Three primer pairs (V1F HRM a V1R-HRM, poxcDNAFw a poxPromRVC, poxcDNAFw a poxPromRVT) were evaluated as the most suitable for distinguishing Lactobacillus bacterial strains.
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Chan, Ming-yan y 陳明恩. "Rapid diagnosis of isoniazid resistant mycobacterium tuberculosis by high resolution melting (HRM) assay". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hub.hku.hk/bib/B48333402.
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Mycobacterium tuberculosis (MTB) is a major infective agent causing human tuberculosis (TB) in the worldwide. Although tuberculosis can be treated by a six-month course of antibiotics, the prevalence of extensively drug-resistance TB (XDR-TB) made the disease becomes a global health problem. In addition to the conventional MTB detection methods, molecular methods become significant in drug resistant MTB detection which can enhance effective drug treatment.
In this study, 200 MTB respiratory specimens were collected from patients with suspected tuberculosis in Tuen Mun Hospital in Hong Kong. Based on the culture method as a gold standard for MTB detection, the presence of MTB in clinical samples was determined by IS6110single tube nested real-time PCR. In addition, by using High Resolution Melting (HRM) analysis, the presence of mutant type KatG315 gene for detecting isoniazid resistant MTB was determined.
Among 66 MTB culture positive samples, 10 samples had positive acid fast bacilli (AFB) smears giving the diagnostic sensitivity 15.1%. IS6110 single tube nested PCR was amplified in 51 specimens giving 77.2% MTB detection sensitivity and 97.8% specificity. Among 51 samples positive for IS6110 PCR, 66.7% showed successful amplification in subsequent KatG-HRM assay. Two samples were confirmed to be isoniazid (INH) resistance in Public Health Laboratory Centre (PHLC). However, there was only one sample showing detectable KatG315 mutation in clinical specimen by using HRM while the other was only detected in the corresponding culture isolate.
From the result of this study, single tube nested real-time PCR demonstrated MTB detection in clinical samples and INH resistant strain with KatG315mutationcan be detected by HRM analysis. Early detection of mycobacteria allow earlier treatment of the patient, thus transmission of the disease can be controlled.published_or_final_version
Microbiology
Master
Master of Medical Sciences
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Cury, Nathália Moreno. "Investigação de Mutações no Gene BRCA1 em Famílias Brasileiras com Suspeita da Síndrome Hereditária do Câncer de Mama e/ou Ovário". Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/17/17135/tde-14062012-134410/.
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Cerca de 10% dos casos de câncer de mama e/ou ovário são caracterizados como hereditários, onde a presença de mutações germinativas no gene de suscetibilidade BRCA1 aumenta o risco de desenvolver esses cânceres durante a vida da mulher. O BRCA1 é um gene supressor tumoral envolvido na resposta de danos ao DNA, controle do ciclo celular, na remodelação da cromatina, ubiquitinação e regulação da transcrição. O presente estudo tem como objetivo central caracterizar as mutações do gene BRCA1 associadas a Síndrome Hereditária do Câncer de Mama e/ou Ovário (HBOC) em pacientes atendidos no Serviço de Aconselhamento Genético do Câncer do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (HCFMRP/USP). Os vinte e dois éxons codificantes do BRCA1 foram analisados utilizando o método de High Resolution Melting (HRM) para triagem de mutações pontuais, seguido pelo sequenciamento de DNA dos casos selecionados para validação. A técnica de MLPA (Multiplex Ligation-dependent Probe Amplification) também foi usada para detectar grandes deleções e duplicações. Uma vez confirmada a mutação, membros da família considerados de alto risco, serão investigados para a mutação específica, a fim de proporcionar-lhes um aconselhamento genético apropriado para a detecção precoce do câncer. No presente estudo, foram investigados 41 pacientes que preencheram os critérios para o teste genético de acordo com NCCN Clinical Practice Guidelines in Oncology v.1.2010. Um total de 21 mutações foram identificadas, duas das quais são patogênicas: a deleção dos éxons 17-18 e a deleção dos éxon 19. Ambas estão localizadas no domínio BRCT do gene BRCA1, essencial para a ligação de fosfoproteínas críticas para a ativação do complexo de reparo do DNA. Outra mutação, a S616del, foi tratada como patogênica, mas apresenta informações controversas em diferentes estudos. O trabalho também identificou uma nova mutação, Val1117Ile. Um estudo de haplótipos das mutações identificadas nos pacientes foi realizado e revelou que um dos haplótipos, denominado de 6, contendo quatro resíduos mutados (871Leu, 1038Gly, 1183Arg e 1613Gly) estava presente em 50% das pacientes. O estudo de associação com 82 indivíduos saudáveis, mostrou diferença significativa (p=0,026) nos pacientes, sugerindo assim um risco aumentado de HBOC. Adicionalmente, foi analisada a mutação germinativa R337H no gene p53 para os casos suspeitos de Síndrome de Li-Fraumeni. Em síntese, o presente estudo contribui com a identificação de uma nova mutação não-sinônina no gene BRCA1 e sugere que o haplótipo 871Leu-1038Gly-1183Arg-1613Gly possa conferir risco aumentado do câncer de mama e/ou ovário em pacientes diagnosticados com HBOC.About 10% of cases of breast and/or ovary cancer are characterized as hereditary, where the presence of germline mutations in susceptibility BRCA1 gene increases the risk of developing these cancers during womans lifetime. BRCA1 is a tumor suppressor gene involved in DNA damage response, cell cycle control, chromatin remodeling, ubiquitination and transcriptional regulation. The present study aims to characterize BRCA1 gene mutations associated with Hereditary Breast/Ovary Cancer Syndrome (HBOC) in patients from the Cancer Genetic Counseling Service of the General Hospital of the Medical School of Ribeirão Preto, University of São Paulo (HCFMRP-USP). The twenty two coding exons of BRCA1 were analyzed using High Resolution Melting (HRM) method for the screening of point mutations, followed by DNA sequencing of the cases selected to validation. MLPA (Multiplex Ligation-dependent Probe Amplification) technique was also used to detect gross deletions and duplications. Once confirmed the mutation, family members most at risk will be analyzed for the specific mutation in order to provide them with an appropriate genetic counseling for early detection of cancer. In the present study, we investigated 41 patients that fulfilled the criteria for genetic testing according to NCCN Clinical Practice Guidelines in Oncology v.1.2010. A total of 21 mutations were identified, two of them are pathogenic: a deletion of exons 17-18 and a deletion of exon 19. Both of them are located in the BRCT domain of BRCA1 gene, impairing the binding of essential phosphoproteins critical to the activation of DNA repair complex. Another mutation, S616del, shows controversial information about its pathogenesis in different studies.The present study also describes a new mutation, Val1117Ile. A study of haplotypes of the mutations identified in patients was performed and revealed that one of the haplotypes, called 6, containing four mutated residues (871Leu, 1038Gly, and 1183Arg 1613Gly) was present in 50% of patients. The association study with 82 healthy subjects showed a significant difference (p = 0.026) in patients, thus suggesting an increased risk for HBOC. Additionally, the germline mutation R337H on p53 gene was also analyzed in the present study for suspected cases of Li-Fraumeni Syndrome. In summary, this study contributes to the identification of a new missense mutation in the BRCA1 gene and suggests that the haplotype-871Leu-1038Gly 1183Arg-1613Gly may confer increased risk of breast cancer and / or ovarian cancer in patients diagnosed with HBOC.
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Milazzo, Ruggero. "Doping of germanium by ion-implantation and laser annealing in the melting regime". Doctoral thesis, Università degli studi di Padova, 2015. http://hdl.handle.net/11577/3424114.
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Germanium is the main candidate for replacing silicon in active regions in future complementary metal-oxide transistors due to: (i) its higher mobility of charge carriers that makes it able to attain higher drive current; (ii) the availability of high-k materials, excellent substitutes for its unstable native oxide and (iii) its lower melting point that allows lower processing temperatures. However, a downscaling beyond 15-nm necessarily requires higher doping levels (higher than 1x10^20cm^-3) beyond the solid solubility of most of the dopants. In particular, n-type ultra-shallow junctions (USJs) are the most challenging, because of the lower solid solubility and higher diffusivity of V-group elements in Ge which make the required shallow doping profiles hard to achieve.
For these reasons, laser thermal annealing (LTA) in the melting regime is a promising advanced activation technology of implanted dopants in Ge for USJs formation. In fact, it has a potential capability of increasing the dopant solubility as well as of confining the diffusion processes into the molten layer, whose depth can be controlled by choosing adequate energy density in turn. Thanks to this technique, donor concentration far exceeding the theoretical maximum solid solubility limits after LTA in Ge are reported in literature for implanted phosphorous and antimony, pushing the doping limits for these systems above to 1x10^21cm^-3.
Despite the above encouraging results, melting LTA on Ge doped with arsenic as well as with acceptors was still unexplored. Therefore, the aim of this work was the investigation of the LTA process applied to both the n-type and p-type doping of Ge after arsenic or boron implantation. In particular, experiments on diffusion, contamination, thermal stability, residual strain and clustering were performed in order to study mechanisms influencing the resulting electrical activation.Il germanio è il principale candidato a sostituire il silicio come substrato per i futuri dispositivi elettronici ultra-scalati, poiché: (i) la sua superiore mobilità di portatori di carica consente correnti maggiori; (ii) la possibilità di crescere ossidi alternativi ad alta capacità dielettrica (high-k), consente di aggirare i problemi legati all’ossido nativo e (iii) la sua minore temperatura di fusione lo rende più facilmente processabile. Tuttavia, miniaturizzazioni che soddisfano i futuri nodi tecnologici (in particolare al di sotto dei 15-nm) necessariamente richiedono livelli di drogaggio più alti di 1x10^20cm^-3, oltre cioè le solubilità solide della maggior parte dei droganti. In particolare, le più problematiche sono le giunzioni ultra-sottili (USJ) di tipo-n, date le basse solubilità e le alte diffusività degli elementi del V gruppo in Ge che rendono gli alti livelli di drogaggio richiesti ardui da ottenere. A questo scopo, il laser thermal annealing (LTA) in regime di fusione rappresenta una promettente avanzata tecnologia di attivazione elettrica dei droganti impiantati, data la sua potenziali capacità di aumentare le solubilità dei droganti, conseguente alla rapidissima ricrescita epitassiale da fase liquida (LPER) indotta, e di confinare i processi diffusivi nella regione liquefatta, la quale viene a sua volta controllata, modulando opportunamente la densità d’energia. Grazie a questa tecnica si sono infatti ottenute in Ge concentrazioni attive che superano ampiamente le rispettive solubilità solide, sia di fosforo impiantato che di antimonio dove si è ottenuto l’impressionante record di 1x10^21cm^-3. Nonostante questi incoraggianti risultati, il LTA nel caso di Ge drogato con arsenico o con accettori non è stato ancora sperimentato. Inoltre, quanto si impiantano grandi fluenze di droganti, si ottengono solo parziali attivazioni elettriche e una profonda comprensione della fenomenologia che avviene durante una così estrema LPER è ancora mancante. Perciò, lo scopo di questo lavoro è stato lo studio del processo di LTA applicato sia per drogaggio di tipo-n che –p di Ge dopo l’impianto di arsenico o boro. In particolare si sono svolti esperimenti svolti sulla diffusione, contaminazione, stabilità termica, stato di deformazione residuo e formazione di clusters al fine di studiarne l’influenza sulla attivazione elettrica risultante.
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Handt, Maximilian Johannes Anton [Verfasser], Jörg T. [Gutachter] Epplen y Bianca [Gutachter] Miterski. "Identifizierung von Mutationen im FMR1-Gen mittels Schmelzkurvenanalyse (High-Resolution-Melting) und DNA-Sequenzierung / Maximilian Johannes Anton Handt ; Gutachter: Jörg T. Epplen, Bianca Miterski". Bochum : Ruhr-Universität Bochum, 2017. http://d-nb.info/1125106638/34.
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Bélteky, Johan. "DNA methylations : A comparison of four genes between Red Junglefowl and White Leghorn". Thesis, Linköpings universitet, Molekylär genetik, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-69086.
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Domestication of animals has given rise to a great phenotypic divergence in selected animals and rapidly generated species of animals more accustomed to human contact and social interactions within the species. Previous studies in chickens (Gallus gallus) have managed to find behavioral and adaptive differences between Red Junglefowl (RJF) and White Leghorn (WL), differences inherent to the domestication process. These phenotypic changes could spawn from a variety of different genomic factors, including an epigenetic gene expression regulatory mechanism known as CpG methylation, a DNA modification of CpG dinucleotides that in turn affect nucleosome formation. In this study we investigated the methylation differences between RJF and WL. This by selecting genes that has previously been shown to be both differentially expressed (DE) and differentially methylated (DM) between RJF and WL, and had shown the same kind of differences in both parental animals and their offspring. By using methylation-sensitive high-resolution melting (MSHRM) we tried to confirm previous DM result, and four genes; FUCA1, RUFY3, PCDHAC1 and TXNDC16 were tested and verified to be DM between RJF and WL.
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Mahapatra, Sailendra Nath. "Determination of Heterogeneity by High-Resolution Seismic Reservoir Characterization in the Heavy Oil Temblor Reservoir of Coalinga Field, California". Diss., Virginia Tech, 2005. http://hdl.handle.net/10919/29377.
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The research focuses on analysis and subsurface imaging of siliciclastics rocks on steam-affected 3D poststack seismic data, merged from different vintages, from the Temblor Formation in the Coalinga heavy oil reservoir in the San Joaquin basin, California. The objective was identification, delineation, and demarcation of reservoir heterogeneities by seismostratigraphic and seismogeomorphic analysis.
The proximity of the San Andreas Transforms greatly controlled basin evolution and caused substantial reservoir heterogeneity by changing the depositional environment from shallow marine to near-shore fluvial. Moreover, two unconformities dissect the reservoir interval.
The seismic dataset exhibits erratic, distorted reflection strengths and amplitudes caused by steam-injection-aided production. A petrophysical analysis based on Gassmann fluid substitution suggests a 27% P-wave velocity decrease in steam-saturated intervals. Seismic to well log ties were problematic and vexing due to the resulting statics, wavelet changes, and line mismatches. Mapping and flattening on a deeper horizon, however, allowed mapping of the internal unconformities and well ties which were crucial for seismostratigraphic sequence identification.
Visualization of seismic attributes brought out stratification patterns and two distinct, laterally and vertically extensive, porous, and interconnected facies tracts interpreted as incised valley fills and tidal-to-subtidal deposits as evidenced by bright, steam related amplitudes.
Seismic attribute analysis, Geobody Visualization and Interpretation, and structure and isochron maps brought out two prominent channel-systems, recut and restacked in the central part of the area. These deposits were identified on seismic data and correlated to high-gamma coarsening-upward sands on logs and cores. The deeper one, shifting towards SSE with depth, lies between the Base Temblor and Buttonbed unconformities both in the southwestern and northwestern parts of the study area and is scattered in the western-central portion. The shallower one originates in southwestern corner below the Top Temblor unconformity shifts towards ESE-SE with depth, and runs nearly parallel to the Top Temblor unconformity. It cuts across the Valv unconformity in central part creating a channel incision, and follows the Buttonbed unconformity towards the north.
The investigation segmented the reservoir into channels, non-channel bearing, and unconformity-bounded subunits which will allow the operator to improve steam injection and optimize placement of oil producing infill wells.Ph. D.
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Daniels, Rachel Fath. "Genomic Tools Reveal Changing Plasmodium falciparum Populations". Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:10931.
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A new era of malaria eradication programs relies on increased knowledge of the parasite through sequencing of the Plasmodium genome. Programs call for re-orientation at specific epidemiological markers as regions move from control towards pre- and total elimination. However, relatively little is known about the effects of intervention strategies on the parasite population or if the epidemiological cues correspond to effects on the parasite population. We hypothesized that genomic tools could be used to track population changes in Plasmodium falciparum to detect significant shifts as eradication programs apply interventions. Making use of new whole-genome sequencing data as well as GWAS and other studies, we used SNPs as biological markers for regions associated with drug resistance as well as a set of neutral SNPs to identify individual parasites. By utilizing tools developed as proxy for full genomic sequencing of the human pathogen Plasmodium falciparum, we characterized and tracked parasite populations to test for changes over time and between populations. When applied to markers under selection - those associated with reduced antimalarial drug sensitivity - we were able to track migration of resistance-associated mutations in the population and identify new mutations with potential implications for resistance. Using a population genetic analysis toolbox to study changes in neutral allele frequencies in samples from the field, we found significant population changes over time that included restricted effective population size, reduced complexity of infections, and evidence for both clonal and epidemic propagation of parasites.
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Vale, Sílvia Daniela Costa. "Uma abordagem epigenética à determinação da idade de amostras biológicas: potenciais aplicações forenses". Master's thesis, Universidade de Aveiro, 2016. http://hdl.handle.net/10773/22398.
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Mestrado em Biologia Molecular e CelularAo longo do tempo de vida, um processo estocástico influenciado pela hereditariedade, fatores ambientais, estilo de vida e doenças conduz a alterações graduais de biomoléculas ao nível molecular e celular – o envelhecimento. Estas alterações podem auxiliar em investigações forenses na determinação da idade de indivíduos e, respetiva identificação, por métodos morfológicos, bioquímicos e moleculares. Contudo, estes marcadores biológicos possuem baixa precisão na estimativa da idade e inúmeras limitações práticas. Uma das modificações epigenéticas que está amplamente correlacionada com a idade é a metilação do ADN, uma vez que os níveis de metilação globais diminuem ao longo do envelhecimento. Desta forma, foram analisados os padrões de metilação de promotores de genes, no sentido de criar um modelo robusto de previsão da idade. Neste estudo, avaliamos a introdução de uma nova abordagem na análise do estado de metilação de três loci (EDARADD, NPTX2 e TOM1L1) – a High-Resolution melting. Inicialmente, foi realizada uma otimização desta metodologia, pela avaliação dos métodos de extração do ADN e dos kits de conversão testados. Estas etapas revelaram ser fatores-chave para a correta análise dos padrões de metilação por HRM num conjunto de amostras estudadas. Esta metodologia permitiu diferenciar as amostras, segundo o estado de metilação dos referidos marcadores, em grupos etários, revelando alguma imprecisão desta técnica para a estimativa da idade. Por último, foi avaliada a metilação global de ADN genómico de uma pequena amostragem, na qual se comprovou a sua diminuição durante o processo de envelhecimento.
Throughout the lifetime, a stochastic process influenced by heredity, environment, lifestyle and disease leads to gradual alterations of biomolecules at molecular and cellular levels – aging. These changes can aid in forensic investigations to estimate the age of individuals and their identification by morphological, biochemical and molecular methods. However, all of these biomarkers have low precision and practical limitations. One of these epigenetic modifications has been correlated with age is DNA methylation, which global level of methylation decreases as a person ages. Therefore, several studies have analyzed the methylation patterns of gene promoters to create a robust model for predicting age. In this study, we evaluated new approach to the analysis of methylation status of three loci (EDARADD, NPTX2 and TOM1L1) – High-Resolution Melting. Firstly, we performed an optimization of this methodology by evaluation of DNA extraction methods and conversion kits. These steps have proven to be key factors for the proper analysis of methylation patterns in a set of samples by HRM. According to methylation status of three markers, this methodology allowed the differentiation of samples in age groups, revealing some technical inaccuracy to estimate age. Lastly, we evaluated the overall methylation of genomic DNA in a sampling and we testified its decrease during the aging process.
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Jamile, Siham. "Polymorphisms in nuclear hormone receptor pathways in breast cancer". Thesis, Queensland University of Technology, 2017. https://eprints.qut.edu.au/108151/2/Siham_Jamile_Thesis.pdf.
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This project was an effort to investigate the relationship between potentially functional biomarkers in Nuclear Hormone Receptor Pathways and sporadic breast cancer susceptibility. The research involved investigation of nuclear hormone receptor co-activator 1 and 3 genes using different molecular genetics methods, resulting in the ability to test their potential effect on the nuclear hormone receptor pathway in sporadic breast cancer.
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Passaro, M. "COST-EFFECTIVE USE OF MOLECULAR MARKERS IN THE PRACTICAL RESOLUTION OF COMMON HORTICULTURAL CHALLENGES". Doctoral thesis, Università degli Studi di Milano, 2017. http://hdl.handle.net/2434/479449.
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Genetic molecular markers (DNA markers) represent genetic differences between individual organisms or species placed directly into DNA sequence. They are widely used as powerful scientific instruments to accomplish different tasks, from genes mapping to forensic discrimination. The tremendous advance in DNA genotyping tools has lead to the development of impressive high- throughput technologies, such as Next Generation Sequencing platforms, that may revolutionize horticulture research and applications. However the cost of such technologies not always make them the most rationale approach, particularly when working on minor crop species or with large number of samples. The present work aims to the exploring a multi-purpose and cost-effective use of different kinds of molecular markers, for assisting fruit tree plants breeding and valorization. For this scope, three cases of study were presented, spanning from cultivar discrimination and phylogeny reconstruction to marker assisted selection (MAS) for Sharka resistance.
D.NA markers such as SSR and AFLP, were successfully used to discriminate the ‘common’ Chinotto from ‘Chinotto di Savona’, an uninvestigated traditional Citrus species cultivated in Liguria (italy) that is gaining increasing interest for the production of high-quality niche food and beverages. New polymorphisms on candidate genes, that could explain some of observed differences between the two accessions, were suggested.
SSR markers were used for the first time to the large-scale application of MAS on apricot (Prunus armeniaca) to boost the conventional breeding programmes. They were found new resistant breeding selections against the most important viral disease of stone fruits, Sharka, caused by Plum Pox Virus (PPV). Novel candidate accessions were also characterized for PPV-resistance, enriching and complementing the apricot germplasm available for breeding. Moreover the number of significant markers required for this task was reduced from seven to two, decreasing the overall cost, in terms of time and resources, usually required for the conventional breeding programmes.
A further reduction of resources for the application of MAS in apricot was achieved developing new SNP markers linked to Sharka resistance, and able to be screened using fluorescence on Real Time PCR machine with or without High Resolution Melting (HRM) technology.
The performed works demonstrate that the correct choice of molecular instruments together with the implementation of new techniques could easily led to cost-effective, time-saving, and reliable results even without the facility and resources reserved for main crops research and applications.
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Farrugia-Jacamon, Audrey. "Investigations moléculaires dans la mort subite du sujet de moins de 35 ans". Phd thesis, Université de Strasbourg, 2012. http://tel.archives-ouvertes.fr/tel-00804339.
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Les canalopathies cardiaques congénitales constituent la principale hypothèse diagnostique dans les cas de mort subite inexpliquée chez les sujets de moins de 35 ans. Notre travail a eu pour objectif demettre au point une stratégie de détection post-mortem des mutations sur les gènes connus pour être impliqués dans les canalopathies cardiaques, applicable en routine, à partir de la principale source d'ADN post-mortem disponible en France à savoir les prélèvements fixés au formol et inclus en paraffine (FFIP). A partir d'une cohorte de 12 cas, deux techniques de détection de variants génétiques ont été évaluées, une technique de criblage par l'analyse des courbes de fusion haute résolution et une technique de génotypage par spectrométrie de masse MALDI-TOF, respectivement sur le gène KCNQ1 et le gène RyR2. Quelle que soit la technique utilisée, il n'est pas possible de s'affranchir du séquençage de type Sanger afin d'explorer les séquences d'intérêts qui n'ont pu être optimisées avec l'une ou l'autre des méthodes à la fois sur les prélèvements congelés et FFIP. L'arrivée des séquenceurs de nouvelles générations ouvrent ainsi de nouvelles perspectives dans ce domaine.
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Vaňásek, Jakub. "Imunomagnetická separace buněk bakterií mléčného kvašení pomocí magnetických nosičů funkcionalizovaných protilátkou". Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2015. http://www.nusl.cz/ntk/nusl-217122.
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Immunomagnetic separation is based on binding of antibody with antigen, where antibody is bound to magnetic particle. In this thesis there were used particles of magnetic pearl cellulose with antiLactobacillus and antiBifidobacterium antibodies. Immunomagnetic separation method was optimalized and verified for its efficiency and specifity with bacterial and yeast cells. This cells were identified by polymerase chain reaction. Efficiency of immunomagnetic separation was verified on probiotic meat product, where Lactobacillus cells were isolated. With DNA from isolated Lactobacillus cells the high resolution melting was performed. The results show presence of several bacterial strains of Lactobacillus species.
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Chen, Shuhui. "Étude des mutations des gènes KRAS, NRAS, BRAF, PIK3CA, MET et de l’expression des protéines P53 et PTEN et leurs implications cliniques dans le carcinome ovarien de haut grade". Thesis, Université de Lorraine, 2016. http://www.theses.fr/2016LORR0093/document.
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Objectifs: Malgré leur grande hétérogénéité histologique et moléculaire, la prise en charge clinique des carcinomes ovariens de haut-grade (COHG) reste peu variable. Le pronostic sombre de cette pathologie implique un réel besoin des nouvelles thérapies. Au-delà des marqueurs pronostiques histologiques classiques et des enquêtes oncogénétiques, l’objectif de cette étude a consisté à rechercher des cibles moléculaires pharmacologiquement recrutables afin de pouvoir proposer aux patientes un accès à la thérapie innovante et personnalisée. Méthodes: Cette étude a été réalisée chez 53 patientes (pts) (âge moyen 58,9 ans, intervalle 25-87) de COHG histologiquement prouvés dont 45 pts de sous-type séreux. 19 pts ont fait l’objet d’une consultation et d’un test oncogénétique sur la base d’antécédents familiaux / personnel de cancer de sein/ovaire. chez. L’expression de P53 et de PTEN a été évaluée sur des tissus fixés au formol et inclus en paraffine par immunohistochimie. Les mutations somatiques de KRAS, NRAS, BRAF, PIK3CA et MET ont été recherchées par PCR-HRM (Polymerase Chain Reaction High Resolution Melting) puis vérifiées par NGS (Next Generation Sequencing) sur des extraits d'ADN préparés à partir d'échantillons de tumeurs congelés, prélevés au moment du diagnostic. Résultats: Des mutations germinales de BRCA1 / 2 ont été identifiées chez 7 pts, toutes atteintes des carcinomes séreux. Une mutation du gène KRAS (exon 2), 2 mutations du gène NRAS (exon 3), 6 mutations du gène PIK3CA (exon 5, 10 et 21) et 5 mutations du gène MET (exon 14 et 18) ont été identifiées chez les 53 tumeurs par NGS, dont deux mutations du gène NRAS et 2 mutations du gène PIK3CA détectées précédemment par PCR-HRM. Aucun profil mutationnel multiple n’a été retrouvé. La surexpression de P53 et la perte d’expression de PTEN ont été constatées chez 32 sur 53 (60%) et 19 sur 46 (41%) des tumeurs. L’analyse statistique n’a été réalisée que chez le sous-groupe de pts atteintes des carcinomes séreux à cause de l’effectif de l’étude. Avec un suivi médian de 38 mois (intervalle de 6-93), 35 pts ont eu une rechute de la maladie et 25 pts sont décédées. La survie sans progression à 2 ans est 28%, et la survie globale à 5 ans est 37%. La surexpression de P53 a été trouvée associée à une meilleure chimiosensibilité, une meilleure survie sans progression et une meilleure survie globale. Conclusion: Pour des COHG, au-delà des altérations de P53 et PTEN, des anomalies génétiques somatiques concernant les voies de signalisation PI3K et MAPK ne sont pas rares et peuvent être détectées par NGS. L’identification de ces anomalies somatiques pourrait offrir une possibilité des thérapies ciblées innovantes pour les patientes sur la base d’éléments diagnostics moléculairesObjectives: Despite the great histological and molecular heterogeneity, the clinical management of high-grade ovarian carcinoma remains univo-cal. As a major subgroup of ovarian carcinoma, high-grade ovarian carci-nomas (HGOC) need novel therapy. Additionally to conventional histolog-ical prognostic markers and oncogenetic investigations, molecular diag-nostic was performed using PCR-HRM (Polymerase Chain Reaction High Resolution Melting) and NGS (Next Generation Sequencing) to identify "druggable" targets that could provide access to innovative personalized therapy. Methods: This study was performed in 53 patients (pts) (mean age 58.9 years, range 25-87) with histologically proven HGOC of which 45 pts with serous carcinoma. BRCA1/2 germline mutations had been screened in 19 pts with familial/personal history of breast/ovarian cancer justifying on-cogenetic investigations. P53 and PTEN expression was assessed on for-malin fixed paraffin-embedded tissues using immunohistochemistry. So-matic mutations of KRAS, NRAS, BRAF, PIK3CA and MET were screened using PCR-HRM and then confirmed using NGS on DNA extracts from frozen tumor specimens taken at diagnosis. Results: Seven pts had BRCA1 / 2 germline mutations, all had serous carcinomas. One mutation of KRAS (exon 2), 2 mutations of NRAS (exon 3), 6 mutations of PIK3CA (exon 5, 10 and 21) and 5 mutations of MET (exon 14 and 18) were identified using NGS, of which 2 mutations of NRAS and 2 mutations de PIK3CA detected previously by PCR-HRM, no multiple mutation was detected. P53 overexpression and PTEN loss of expression was detected respectively in 32 of 53 (60%) and 19 of 46 (41%) of all the tumors. Because of the efffective of the study, statistical analyses were restricted to pts with serous carcinoma. With a median follow-up of 38 months (range 6-93), 35 pts had disease progression and 25 pts died during the follow-up. The 2-year progression-free survival (PFS) rate was 28% and 5-year overall survival (OS) rate was 37%. Overexpression of mutant P53 was found to be associated with chemosensitivity and longer PFS and OS. Conclusion: In HGOC, beside P53 and PTEN alterations, somatic genetic abnormalities of PI3K and MAPK signaling pathways can be detected us-ing NGS and provide molecular rationale for targeted therapies, potential-ly offering new therapeutic opportunities to the patients
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ER, TZE-KIONG y 余志強. "Molecular Diagnosis of Genetic Diseases by High-Resolution Melting Analysis". Thesis, 2011. http://ndltd.ncl.edu.tw/handle/49092182181899780900.
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博士高雄醫學大學
醫學研究所
100
Identifying and understanding genetic variation between populations and individuals is an important aim in the field of genomics. The unique genetic profile of an individual confers susceptibility to a given trait or disease. Therefore, there is a rapidly growing interest in feasible methods for mutation screening in life science research. High-resolution melting (HRM) analysis represents the next generation of mutation scanning technology and offers considerable time and cost savings prior to other screening method. HRM is a novel, homogeneous, close-tube, post-PCR method, enabling researchers to analyze genetic variations in PCR amplicons. Samples can be discriminated according to their sequence, length, GC content or strand complementarity. Based on its ease of use, simplicity, flexibility, low cost, nondestructive nature, high sensitivity, and specificity, HRM analysis is quickly becoming the tool of choice to screen patients for pathogenic variants. Here we briefly discuss the establishment of HRM analysis for mutation screening including JAK2 V617 missense mutation and ETFDH gene mutation. Taken together, HRM analysis can be used for high-throughput mutation screening for research, as well as for molecular diagnostic and clinical purposes. Janus kinase 2 (JAK2) is a tyrosine kinase involved in the cytokine signaling of several growth factors such as erythropoietin and thrombopoietin in normal and neoplastic cells. The G to T exchange at nucleotide 1849 in exon 14 of the JAK2 gene leads to a substitution of valine with phenylalanine at the amino acid position 617 (V617F) of the JAK2 protein. Currently, the occurrence of the JAK2 V617F mutation is well recognized in myeloproliferative disorders (MPDs), such as essential thrombocytosis, polycythemia vera, and primary myelofibrosis. The identification of molecular lesions specific to the myeloproliferative neoplasms, in particular JAK2 V617F, has broadened understanding of the common features within these disorders and has advanced diagnostic, prognostic, and therapeutic tools. The aim of our study was to assess the value of the HRM analysis using real-time polymerase chain reaction (PCR) (Lightcycler® 480; Roche Applied Science) for identifying the JAK2 V617F missense mutation. Our results showed that up to 5% of the JAK2 V617F mutation was successfully detected in patients with MPD using HRM analysis. The results proved 100% comparable to those obtained by ARMS assay. In conclusion, the HRM analysis is a rapid and effective technique for the detection of JAK2 V617F missense mutation. Multiple acyl-CoA dehydrogenase deficiency (MADD) or glutaric aciduria type II is an autosomal recessive disease caused by defects in mitochondrial electron transfer system and metabolism of fatty acid. Recently, ETFDH mutations were reported to be major causes of riboflavin-responsive MADD. The present study is aimed at screening ETFDH mutations by HRM analysis. Our results showed that HRM analysis proved to be feasible in detecting 3 known (c.250G>A, c.380T>A, c.524G>T) and 1 novel (c.1831G>A) ETFDH mutations. Each mutation could be readily and accurately identified in the difference plot curves. We estimated the carrier frequency of the hotspot mutation, c.250G>A, in the Taiwanese population to be 1:125 (0.8%). In summary, HRM analysis can be successfully applied to screen ETFDH mutations. Since riboflavin-responsive MADD is often treatable, especially with mutations in ETFDH, identifying ETFDH mutations is crucial for these patients. Interestingly, two of the mutations (p.Ala84Thr and p.Phr128Ser) are located in the FAD-binding domain; however, the two amino acids do not have direct interactions with FAD according to the predicted 3D structure of ETF:QO. Therefore, to explore the effects of the mutations on ETF:QO dynamics, molecular dynamics (MD) simulations of the wild type (WT) and mutant type (MT) ETF:QO in the same model environment were compared. Besides the MD simulations, an alternative method, normal mode analysis (NMA), for studying protein motions was used to analyze the dynamic correlations between the mutation sites and the FAD-binding motif. Using molecular dynamics (MD) simulations and normal mode analysis (NMA), we found that the p.Ala84Thr and p.Phe128Ser mutations are most likely to alter the protein structure near the FAD binding site as well as disrupt the stability of the FAD binding required for the activation of ETF:QO. Intriguingly, NMA revealed that several reported disease-causing mutations in the ETF:QO protein show highly correlated motions with the FAD-binding site. Based on the present findings, we conclude that the changes made to the amino acids in ETF:QO are likely to influence the FAD-binding stability.
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Yang, Ching Yuan y 楊晴媛. "Rapid Identification of Clinical Mycobacteria by High-resolution Melting Curve Analysis". Thesis, 2009. http://ndltd.ncl.edu.tw/handle/43286758890797758663.
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碩士長庚大學
醫學生物技術研究所
97
The conventional way for mycobacterial species identification usually requires an experienced personal and is time-consuming. In this study, we explored the combined use of real-time PCR and high-resolution melting (HRM) curve analysis for rapid detection and identification of clinically important mycobacterial species. At first, the HRM profiles for sixteen mycobacterial species, including M. tuberculosis (MTB), M. asiaticum, M. gordonae, M. kansasii, M. szulgai, M. avium, M. fortuitum, M. chelonae, M. intracellulare, M. marinum, M. terrae, M. xenopi, M. scrofulaceum, M. abscessus, M. haemophilum and M. triviale, were established using CAP standard mycobacterial isolates. All these 16 species can be identified by the HRM profiles after heteroduplex formation with M. Kansasii. To further extend the clinical application of this method, 108 clinical mycobacterial isolates were subjected to a blind HRM analysis. The results showed that the species of 91 isolates were correctly identified with the accuracy rate equivalent to 84%. Eight of the isolates were not identified correctly, although all of them can be identified by traditional characteristics of growth rate and pigmentation. In addition, 9 isolates can not be identified due to the failure of PCR. Together, this method is rapid and cost-effective without using probe for detection. Hence, the combined use of real-time PCR and high-resolution melting curve analysis offers a good way for rapid identification of clinical mycobacteria in a clinical setting.
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Lee, Ta Hsien y 李達憲. "Rapid and sensitive human papillomavirus genotyping by high-resolution melting analysis". Thesis, 2010. http://ndltd.ncl.edu.tw/handle/69433311808960630216.
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碩士長庚大學
生物醫學研究所
98
Abstract Human papillomavirus (HPV), a small, non-enveloped, double-stranded DNA virus, is established as the key etiological factor in cervical neoplasm. More than 90% of cervical neoplasm is attributed to HPV infection. Evidence is accumulating that HPV genotyping may be useful for patient management in the future. To establish a fast and cost-effective high-resolution melting (HRM) assay system for the detection of the ten clinically most relevant high-risk HPV (HR-HPV) types. All procedures of HRM differential system are finished in single machine and cost only 3 hours including the operating procedures, and we can perform more than 300 specimen samples at same time. Besides, HRM is a high sensitive method that the detective limitation is between 30-300 HPV copies. Till now, we have successful differentiated 10 HR-HPV genotypes (HPV16, 18, 33, 39, 45, 51, 52, 56, 58 and 68) which represent over 90％ clinical cases in Taiwan by the normalized melting curve and derivative plot of HRM. Each genotype contains the specific curve in two HRM plots. As for these 10 HR-HPV, the characteristics of HPV18, 39, 45 and 51 are un-clearer than the others. Therefore, we attempt to add the unlabeled oligoantisense (unlabeled probe) to induce the other special signal so that we can differentiate them from all HR-HPV easier. And we can detect HPV variants with the addition of unlabeled probe. Finally, we have applied this differential system on clinical sample test. And the other 3 HR-HPV genotypes will be found in the following test.
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Hsu, Wei-Ting y 徐瑋廷. "Rapid Identification of Allergenic Fungi Using High-Resolution Melting (HRM) Analysis". Thesis, 2015. http://ndltd.ncl.edu.tw/handle/59355858603715680842.
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碩士輔仁大學
生命科學系碩士班
104
In our living environment, excess amounts of airborne fungal spores, especially for the existences of allergic fungi, might have adverse effects on human health as the quality of air deteriorates. It is urgent to build a detection and identification platform and to assess the health risks and hazards for these allergic fungi. Traditionally, fungi are detedted and classfied mainly by their morphological characteristics and biochemical features, among others. This research is intended to employ the melting profiles as molecular fingerprints for fungal species detection and identification to assist the disease management of allergic fungi. High-resolution melting (HRM) analysis, compared with the traditional agarose gel electrophoresis, is rapid, cost- effective and more powerful in resolving the difference in the DNA sequences of interest. Totally, 10 allergic fungal strains, belonging to 7 genera, prevalent in Taiwan including BCRC 31123, BCRC 30812, BCRC 32888, BCRC 32054, BCRC 30099, BCRC 30473, BCRC 34548, BCRC 30010, BCRC 32490 and BCRC 32628 as well as HR-1 instrument were used. Six universal primer sets ITS1/ITS4, 28SU1/28U2, IGSV3/ IGSV4, Ascoll1/Ascoll2, 1a-F2/Ascoll2 and LR3R/LR7 were chosen to amplify the 5.8S, LSU, ITS, and IGS rDNA fragments, respectively. The results showed that the HRM profiles(-dF/dT profiles) of the LCGreen+ -labelled ITS or LSU amplicons, when primer ITS1/ITS4 or LR3R/LR7 was used, the resulting distinguishable profiles could be classified into 10 types. When primer sets ITS1/ITS4, 28SU1/28U2 and LR3R/LR7 were subsequently used in pairs for the Duplex PCR studies, the results indicated that the ITS-LSU amplicons, if primers ITS1/ITS4 and LR3R/LR7 were used, resulted in distinctive melting profiles and could group the examined strains into 10 types. In addition, the specificity was evidently higher than those of the Singlex PCR. Lastly, the result proved that the Multiplex PCR, with the primers ITS1/ITS4, 28SU1/28SU2 and LR3R/LR7, coupled with the HRM analysis was the most useful mean to differentiate the 10 examined strains.
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Lin, Jiunn-Yow y 林俊佑. "Rapid Identification of Bacterial Food Poisoning Pathogens by High-Resolution Melting Curve Analysis". Thesis, 2013. http://ndltd.ncl.edu.tw/handle/24203145926023478068.
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碩士輔仁大學
生命科學系碩士班
101
Foodborne illness is one of the most common infectious diseases worldwide. Among them, food poisoning syndromes caused by bacterial pathogens are known to be very diversified and complicated. Therefore, how to clarify food poisoning pathogens as soon as possible is an important issue. The conventional methods used for the diagnosis of bacterial food poisoning pathogens are very time-consuming. However, with the advance of cross-cutting diagnostic technology, many new tools are available for an accurate and rapid bacterial detection and diagnosis. Among them, nucleic acid-based detection and diagnosis is one of the most promising sectors in in vitro diagnostics. In this study, a high-resolution melting curve method (HRM) was employed for developing the detection and identification methods of the above- mentioned pathogens. Totally, 7 genera of DNA samples, including Bacillus spp., Staphylococcus spp., Escherichia spp., Salmonella spp., Shigella spp., Pseudomonas spp. and Vibrio spp. were used. The ribosomal DNA sequences of each species were retrieved from the GenBank and aligned, the primers for polymerase chain reaction (PCR) was subsequently designed. The HRM profiles of the 16S rDNA and ITS (internal transcribed spacer) amplicons were examined to characterize inter-species and inter-subspecies difference. The amplicons of the 16S rDNA variable regions were obtained lastly with the hn-PCR(hemi-nested PCR) protocol, and resolved then by the HRM analyses. The results have clearly demonstrated that the HRM profiles revealed by the aforementioned approach could differentiate at least 7 species of the 5 different genera. Moreover, it was capable of resolving the difference at the subspecies level. Additionally, the phylogenetic relationship reconstructed using the neighbor-joining method, based on the 16S rDNA sequences of the examined species, was also shown that the closer the phylogenetic relationship, the similar the HRM profile. The potential application and prospect of this study as well as the plausible approach to build an identification and diagnostic platform were addressed finally.
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Chen, Guan-Hong y 陳冠宏. "Study on the analysis of oral microbes by High-resolution melting (HRM) technology". Thesis, 2016. http://ndltd.ncl.edu.tw/handle/58668405382362309211.
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碩士輔仁大學
生命科學系碩士班
104
The association of two oral diseases, dental caries and periodontal disease, and microbial communities present in human oral cavities has been well-documented for decades. However, a fast and powerful detection, identification platform for assessing oral microbes is needed for an effective disease management of above-mentioned diseases. The aim of this research is to develop a rapid, simple yet reliable, cost-effective screening method using the HRM(High-Resolution Melting) technology. The HRM technique, basing on detecting small differences in PCR melting curves, resolves small sequence variations in DNA samples examined. Totally 62 bacterial isolates from 71 subjects were studied. The level of fluorescence versus temperature of the fluorescent dye LC Green+-labeled 16S and 23S amplicons were real-timely monitored using the HR-1 instrument to generate the melting curves for subsequent assessment of sequence variations. Eleven groups, providing genus-level identification, could be classified by using the HRM profiles of the 16S fragments amplified from 62 isolates with the 16S universal primers 8F/1492R(1), together with the molecular taxonomic information of the 24 representative isolates. To increase the sensitivity, the HRM profiles of 24 isolates were further examined with the shorter DNA fragments amplified with primer sets 178165/178163(for 16S amplicon) and 1782352/1782332(for 23S amplicon), respectively. The results revealed that the HRM profiles of the 16S amplicons were more powerful in clustering examined isolates than those of 23S. Regarding the molecular typing, although the conclusion remained unchanged, their HRM profiles were in general more characteristic than the previous ones. Lastly, to mimick the mixed composition of microbes normally found in field samples, different known DNA samples were mixed and analyzed with the PCR-HRM protocol, the results indicated that the current protocol was incapable of effectively distinguishing the component species in all cases.
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Lin, Pin-Rung y 林品瑢. "Rapid detection of common food-poisoning bacterial pathogens by high-resolution melting analysis". Thesis, 2016. http://ndltd.ncl.edu.tw/handle/80950573423772084630.
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碩士輔仁大學
生命科學系碩士班
104
The World Health Organization had estimated that approximately two billion foodborne illness outbreaks from 1990 to 2010 caused 0.89 to 1.4 million deaths worldwide. In Taiwan, the main cause of foodborne diseases is resulted from ingestion of foodstuffs contaminated with bacteria and, therefore, how to rapidly detect and identify food-posioning bacteria is an important public health problem. Bacteria are classified traditionally using cultural characteristics and biochemical features, among others. This study, aiming to develop a technical platform providing a low cost, rapid yet sensitive method for detecting and identifying the lab pure samples and the on-site mixed samples, has evaluated multiple biomarkers and optimized the detection protocol based on the HRM (high-resolution melting) technique. Totally 19 primers were used to amplify sequences of five biomarkers, namely, EF-G (elongation factor G), EF-Tu (elongation factor thermo unstable), rpoB (beta subunit of RNA polymerase), sodA (superoxide dismutase A) and FtsZ (filamenting temperature-sensitive mutant Z), from seven species (Staphylococcus epidermidis, Staphylococcus aureus subsp. aureus, Escherichia coli, Shigella sonnei, Salmonella enterica subsp. arizonae, Salmonella enterica subsp. enterica, and Bacillus cereus) using traditional PCR and hn-PCR(hemi-nested PCR). The PCR amplicons were separated by agarose gel electrophoresis (AGE), and the LCGreen plus labeled amplicons were analyzed by the HRM techniques. For every biomarker, the melting curve (-dF/dT) was more powerful in resolving the molecular difference among species examined than the AGE method. The biomarkers could be selected for effective detection varied among bacterial species. Basing on the melting curves of PCR amplicons resulting in amplifications of traditional PCR, biomarker EF-G could be used to specifically detect Bacillus strain. sodA could be used to separate two species (B. cereus and S. enteric). rpoB could be employed to differentiate five species including B. cereus、S. enteric,S. aureus,S. epidermidis and E. coli. Lastly, EF-Tu and FtsZ could be used for differentiation of all examined species. Furthermore, the melting curves of EF-Tu(tsh) amplicons obtained from the hn-PCR protocol could be employed to distinguish B. cereus,S. epidermidis and E. coli. The melting curves of rpoB-ba and rpoB-sta.a amplicons could be used to specifically identify B. cereus and S. aureus, respectively, while those of FtsZ-bs amplicons could be use to specifically detect S. aureus. To evaluate the potential of HRM technique in on-site detection for rapidly and sensitively screening multiple targets at the same time, the amplicons of various biomarkers resulting in amplifications of traditional PCR and hn-PCR from the DNA mixture solutions (comprising of different DNA targets and different proportion of component DNA templates) were used for subsequent HRM analysis. For every available amplicons it was impossible to identify the component species form the resulting melting curves using the current protocol.
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Chang, Chi-Yuan y 張啟源. "Analysis of cytochrome P450 2A6 polymorphism in oral cancer by High Resolution Melting". Thesis, 2011. http://ndltd.ncl.edu.tw/handle/58300457088035070366.
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碩士高雄醫學大學
牙醫學研究所
99
ACKGROUND:CYP is now widely known as a family of enzymes metabolizing a wide variety of xenobiotics, including drugs and carcinogens. Human cytochrome P450 2A6(CYP2A6) is an important member of the CYPs and CYP2A6 has been reported that associated with risk of cancer Oral squamous cell carcinoma(OSCC) is one of the high lethality in Taiwan, to discover the cancer appear at the early year, we extremely need a fast, convenient and cheap way to screen OSCC. High Resolution Melting Analysis(HRMA) detect and distinguish wild type homozygote, heterozygote and mutative homozygote by fluorescence differ at the melting temperature . Material and Method:DNA sample from peripheral blood of 60 normal men and 55 OSCC patient/men, analysis the genotype and allele frequency by Q-PCR and HRMA. This is the first time to to analyze the CYP2A6 polymorphism by HRMA. Results:Genotype of CYP2A6, CYP2A6*1A/CYP2A6*1A, CYP2A6*1A/CYP2A6*1B, CYP2A6*1A/CYP2A6*5, CYP2A6*1A/CYP2A6*7, CYP2A6*1A/CYP2A6*9, CYP2A6*1B/CYP2A6*1B, CYP2A6*1B/CYP2A6*5, CYP2A6*1B/CYP2A6*7, CYP2A6*1B/CYP2A6*9, CYP2A6*5/CYP2A6*9, CYP2A6*9/CYP2A6*9, and allele of CYP2A6 were deteced. The frequency with which the subjects carried homozygotes of the CYP2A6* 9 which causes lack of the enzyme activity, was lower in the OSCC patients than in the healthy control subjects. The odds ratio (OR) of the group homozygous for the CYP2A6* 9 was significantly lower and calculated to be 0.08 (95% CI; 0.01–0.77) when the OR for the population with homozygotes ofthe CYP2A6 wild-type gene was defined as 1.00. In the allelic-base analysis, there was also a significant decrease in the OR for the CYP2A6*9 allele. Conclusion: A new and fast way to detect the group with high risk of OSCC and we suggest that CYP2A6* 9/ CYP2A6* 9 play an important role to decrease the risk of OSCC
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Tseng, Li-Ping y 曾麗憑. "Association of Breast Cancer with Six Known SNPs by High-Resolution Melting Analysis". Thesis, 2011. http://ndltd.ncl.edu.tw/handle/73370248602625660620.
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碩士高雄醫學大學
醫學研究所
99
Genome Wide Association Study(GWAS) is an examination of all or most of the genes of different individuals of a particular species to see how much the genes vary from individual to individual. Different variations are then associated with different traits, such as diseases.We reffer GWAS results and choose 6 known SNPs related with breast cancer including Fibroblast growth factor receptor-2(FGFR2) rs1219648 and rs2420946、TNRC9 (Trinucleotide repeat containing 9) rs3803662、2q35 rs13387042、8q24 rs10505477、and Reelin (RELN) rs17157903 to examination by High Resolution Melting Analysis (HRM),and try to find out the association of Taiwan breast cancer with thease six known SNPs . SNP in different ethnicities has different associated .For example, FGFR2 rs1219648 and rs2420946 had significant risk with breast cancer for African -American women and Caucasian women；The other Genome Wide Association Studies(GWAS) discovered RELN rs17157903、2q35 rs13387042 and TNRC9 rs3803662 had significant association with breast cancer for European or American women .In our study ,none of thease five SNPs had significant association with breast cancer for Taiwan women. However, the 8q24 rs10505477genotype TT showed a significant association with breast cancer compared with general genotype CC (OR＝0.65 ; 95% CI＝0.43，0.98; P＝0.041), allele frequency among the 8q24 rs10505477 also showed a significant association with the breast cancer （OR＝0.76 ; 95% CI＝0.65，0.98; P＝0.031）.Moreover , 8q24rs10505477showed significant association with the clinical characteristics of Estrogen receptor（ER）and Progesterone receptor（PR）.
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Su, Yu-Ling y 蘇俞綾. "Detection of DNA Methylation Levels in Oral Cancers Using Methylation-sensitive High-resolution Melting Analysis". Thesis, 2010. http://ndltd.ncl.edu.tw/handle/92749224243985977546.
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碩士中國醫藥大學
醫學檢驗生物技術學系碩士班
98
DNA methylation plays an important role in the process of gene transcription and regulation. It has been reported that aberrant methylation renders the occurrence of cancer, including oral cancer. Methylation-Specific PCR (MS-PCR) followed by HRM analysis (MS-HRM) provides a highly sensitive method for the detection of low level methylation and quantification of methylated DNA. The object of this study is to establish the MS-HRM method to detect the DNA methylated level in clinical oral cancer samples. The detection limitation is 1.25 ng of bisulfite genomic DNA. The clinical oral cancer tissues were collected from the patients whose progressions have been well diagnosed and staged. One hyper-methylated and one hypo-methylated candidate gene which previously identified by CpG island microarray were selected to detect the methylation level in clinical oral cancer tissues by MS-HRM method. For hyper-methylated gene, RARB, the frequency of the gene methylation in oral cancer tissue was significantly higher than that in normal tissues. Moreover, the gene methylation status is associated with early step of tumor progression. On the other hand, the hypo-methylated gene, PTHrP, the frequency of the gene methylation in oral cancer tissue was significantly lower than that in normal tissues. Moreover, the gene methylation status is associated with tumor-node-metastasis staging in oral cancer samples. Taken together, we develop a sensitive and quantitative method to detect DNA methylation level in clinical sample. Our results may shed a light to understand the pathogenesis of oral cancer and assistant advanced in clinical diagnosis for oral cancer.
46
Huang, Shao Tung y 黃少東. "Methylation Quantification of Tumor Suppressor Genes in NPC by Methylation-Sensitive High Resolution Melting Analysis". Thesis, 2012. http://ndltd.ncl.edu.tw/handle/89909914537481784618.
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碩士長庚大學
生物醫學研究所
100
Aberrant DNA hypermethylation of tumor suppressor genes (TSGs) inhibits gene expression and causes tumor progression. Since TSGs hypermethylation often used as biomarkers reflecting tumor progression, we used methylation specific high resolution melting (MS-HRM) PCR-based analysis to rapidly detect DNA methyation percentage of aberrant hypermethylated genes in nasopharyngeal carcinoma (NPC). We previously identified a novel hypermethylated gene, Gene A, in NPC biopsies. Gene A is a transcription factor which regulates body axes development. According to our bisulfite sequencing data , Gene A was hypermethylated in NPC cell lines and NPC tumor biopsies (65~95%), however, Gene A gene of adjacent normal tissues and normal individuals’ white blood cells was hypomethylated (8~65%). To test whether MS-HRM has comparable results as bisulfite sequencing, we performed MS-HRM using the same samples. Indeed, MS-HRM had consistent results as bisulfite sequencing. Besides, MS-HRM is a more rapid and economic analysis when compared with bisulfite sequencing . Since EBV copy number is considered as prognostic marker for NPC, we isolated 27 cell-free DNA samples from 5 NPC patients’ plasma at difference time. We further detected EBV copy number and Gene A methylation, respectively. Our results indicated that EBV copy number was positively correlated with Gene A methylation . Hence, the copy number of EBV in plasma could reflect Gene A methylation level in tumor. Taken together, we have developed a rapid and accurate MS-HRM method to detect Gene A hypermethylation; we may use this gene as a biomarker for NPC.
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BRACALINI, MATTEO. "Understanding Alien Pests: the Challenge of Complementary Research on Dryocosmus kuriphilus and Leptoglossus occidentalis in Italy". Doctoral thesis, 2015. http://hdl.handle.net/2158/957158.
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The results presented in this dissertation allow to better understand the outbreak dynamics of the ACGW, as well as the importance of different chestnut cultivar susceptibility. Furthermore, studying the role of native parasitoids is crucial to assess the impact of T. sinensis on endemic ACGW natural enemies once the exotic parasitoid colonizes these areas, either naturally or by future introductions. As regards the WCSB two highly specific DNA-based diagnostic protocols were devised, showing a promising sensitivity in the detection of WCSB biological traces. In addition, the potential of HRM analysis for insect genotyping was highlighted.
48
Hsieh, Li-Ling y 謝麗鈴. "High-Resolution Melting Analysis for Rapid Detection of BRAF and PIK3CA Gene Mutations in Colorectal Cancer". Thesis, 2011. http://ndltd.ncl.edu.tw/handle/47701699472255969206.
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碩士高雄醫學大學
醫學研究所
99
Epidermal growth factor receptor (EGFR) monoclonal antibody therapy is established in patients with wild-type KRAS colorectal cancer ; however, up to 50% of these patients do not respond to this therapy. To identify the possible causes of this treatment failure, we searched for mutations of several oncogenes involving in the EGFR-dependent signaling pathways. In this study, high resolution melting analysis (HRMA) was used to screen hot-spot mutations in the BRAF and PIK3CA genes in 182 colorectal cancer specimens. Direct sequencing was used to confirm HRMA results. Activating mutations were detected in 28.6% of KRAS (codon 12, 13), 1.1% of BRAF (V600E), 4.9% of PIK3CA (exon 9, 20) and 4.9% (KRAS and PIK3CA) of the 182 colorectal cancer specimens. HRMA provides a fast, high sensitivity, and valid approach to efficiently detect these oncogene mutations. Failure of EGFR antibody therapy in patients with wild-type KRAS colorectal cancer may result from activating BRAF or PIK3CA mutations.
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CHEN, YI PING y 陳怡蘋. "Analysis of extended spectrum β-lactamasesKlebsiella pneumoniae blaSHV gene codon 238and 240 by high-resolution melting method". Thesis, 2012. http://ndltd.ncl.edu.tw/handle/07642309211452120941.
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碩士高雄醫學大學
醫學研究所
100
The aim of this study was to investigate the blaSHV gene codon 238 and 240 polymorphisms in extended-spectrum beta-lactamase(ESBL) Klebsiella pneumoniae. In this study, the high-resolution melting (HRM) and DNA sequencing were used as the research tools. In the technology of HRM, the PCR products and the saturated DNA dye were used to record the result of the test of the sample by using the high-resolution melting curves.The blaSHV gene was isolated from ESBL- Klebsiella pneumoniae at Kaohsiung Municipal Hsiaokang Hospital from January, 2010 to December, 2011. Among 114 isolated ESBL-KP, there were 85strains (74.56％) wild type ( GGC- GAG ) mutant, 3 strains (2.63％) codon 238 G238S ( GGC→AGC) mutant, 4 strains (3.51％) codon 240 E240K ( GAG→AAG) mutant, and 22 strains (19.30％) codon 238 and 240 (GGC- GAG →AGC- AAG ) with double mutants. The technology has 100.0% of sensitivity, 100.0% of specificity, 100.0% of positive predictive value, and 100.0% of negative predictive value.We can conclude that in molecular epidemiological studies, the high-resolution melting (HRM) is a good identification tool for bacteria genotyping.
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Chang, Shy Shin y 張詩鑫. "Detection of bacterial infection by the combined use of real-time PE-PCR and high-resolution melting analysis". Thesis, 2015. http://ndltd.ncl.edu.tw/handle/08105975222447250420.
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博士長庚大學
臨床醫學研究所
103
Sepsis remains to be the leading cause of mortality in critical care patients. Early identification of causative pathogen in sepsis patients can improve clinical outcome, and the current gold standard is to use blood culture. However, blood culture is often not effective in identifying the pathogens in three common types of sepsis patients: (I) patients that have been recently treated with antibiotics before blood culture; (II) patients that have cytokine disorder instead of microbial infection; (III) patients that are infected with pathogens that are not easily cultured. Even if blood culture can identify the causative pathogen, it is rather time consuming, and often requires 48-72 hours to identify the microbial. Due to the above problems, clinicians often rely on empirical antibiotic treatment modalities for sepsis patients. This is because the risk of mortality increases substantially in hourly increment when the appropriate antimicrobial therapy is delayed. Although use of empirical antibiotic can be effective, it can instead cause an emergence of drug-resistant organisms. Thus, my goal is to establish a rapid effective diagnostic tool for bloodstream infections, and thereby help clinicians select the most appropriate antibiotic treatment for sepsis patients. My research consists of two different parts. The first part is the establishment of a new innovative molecular diagnostic technique for microbial identification. To quantitatively identify microbial, I have combined real-time polymerase chain reaction (PCR) and high-resolution melting (HRM) technology. Using slightly different approaches, I have successfully identified 25 clinical common pathogens using this platform: 9 bacterial species can be identified via a 1-step post-PCR high-resolution melting analysis; 12 bacterial species can be identified via the high-resolution melting plots obtained by heteroduplex formation between the PCR products of the tested and reference bacterial species; and 4 bacterial species can be identified by a 2nd real-time PCR targeting a different region of the 16S ribosomal ribonucleic acid (rRNA) gene. The second part of my thesis is to solve bacterial deoxyribonucleic acid (DNA) contamination in PCR reagents. To solve contamination, we have employed broad-range primer extension-PCR (PE-PCR) strategy that obviates the need for DNA decontamination. Broad-range PE-PCR amplification of the 16S rRNA gene can be validated and minute quantities of template DNA (10 femtogram) was detectable without false positives.