Dissertations / Theses on the topic 'PCR-based diagnostic'

Salmonellae are enteric bacteria infecting animals and humans. Large animal clinics and Veterinary Teaching Hospitals are greatly affected by Salmonella outbreaks and nosocomial infection. The risk of environmental contamination and spread of infection is increased when animals are confined in close contact with each other and subjected to increased stress factors. This study was designed to compare double-enrichment culture techniques with Gel-based and Real-time PCR assays in the quest for improved diagnostic methods for detecting Salmonella in equine fecal samples. 120 fecal samples submitted to the Clinical Microbiology Laboratory of the Veterinary Medical Teaching Hospital at Texas A&M University (CML, VMTH, TAMU) were tested for Salmonella using all three techniques. Double-enrichment bacterial culture detected 29 positive results (24%), Real-time PCR detected 33 positive results (27.5%), and Gel-based PCR detected 73 positives results (60.8%). While culture and real-time PCR methods had similar results, the gel-based PCR method detected many more positive results, indicating probable amplicon contamination. Real-time PCR can be completed as soon as the day after submission while culture techniques may take 2 to 5 days to complete. However, viable bacterial cells are needed for antimicrobial susceptibility testing and serotyping: both important for epidemiological studies. Therefore, double-enrichment bacterial culture performed concurrently with real-time PCR methods could be efficient in clinical settings where both accurate and expedient results are required.

Thesis (Ph.D.) - University of Glasgow, 2007.
Ph.D. thesis submitted to the Division of Community Based Sciences, Faculty of Medicine, University of Glasgow, 2007. Includes bibliographical references. Print version also available.

Outbreaks and sporadic cases of viral Infectious Intestinal Disease (IID) are a major public health issue resulting in significant morbidity and sometimes mortality each year. The economic costs associated are substantial. Laboratory diagnosis of viral IID is important as the many infectious and non-infectious causes cannot be reliably differentiated using clinical or epidemiological characteristics alone. An accurate diagnosis can aid patient management, infection control procedures and reduce health care costs by preventing unnecessary treatments, testing for alternative causes and hospital stay. It also aids public health surveillance. At the start of the research described in this thesis the West of Scotland Specialist Virology Centre (WOSSVC) used Electron Microscopy (EM) as the frontline test for outbreaks and sporadic cases of IID. However, although rapid on a small number of samples, this technique has been shown to be insensitive, laborious and is not suited to testing large numbers of samples. The research presented in this thesis sought to examine whether molecular diagnostic techniques such as conventional gel-based or real-time Polymerase Chain Reaction (PCR) assays could be a viable replacement for EM as the frontline test(s) for viral IID in a routine laboratory service of this type, and whether their implementation could bring benefits to the laboratory service in terms of improved rapidity, sensitivity and throughput. The aim was to adapt published PCR methods for use in routine diagnostic work rather than for research purposes, an approach that distinguishes this research from previous work in this area. In order to achieve this aim, the appropriate PCR techniques were first selected from the literature, based on a combination of clinical and laboratory requirements, and were adapted for use in the laboratory service. A series of laboratory experiments was then carried out in order to compare the sensitivity of the adapted methods to existing techniques such as EM and antigen detection assays (EIAs) and to other methods that emerged during the period of study including alternative PCR assays. Where found to be suitable, the selected PCR tests were implemented in the routine diagnostic service for viral IID. The effects of these changes on the laboratory service were then examined. The results show that since the introduction of molecular tests at WOSSVC for the detection of viral pathogens in cases of gastroenteritis the number of samples tested has risen steadily, as have the detection rates for each of the main viral causes of IID. Furthermore, this has been achieved at the same time as a substantial reduction in sample turn-around-times. Such improvements will have a positive impact in several areas of public health relating to viral IID and are discussed fully, including patient management, infection control and national surveillance.

In this study we present a poly-specific PCR-based, high-throughput sequencing (HTS) diagnostic system together with an appropriate data analysis pipeline for the diagnosis of grapevine viruses. Poly-specific and virus-specific primers were established to be capable of detecting and identifying 37 grapevine infecting viruses from 11 genera. An analysis pipeline using CLC Genomics workbench was developed by utilising various defined artificial samples which were assembled and sequenced on the Illumina MiSeq platform. A threshold for percentage mapped reads of 0.4% during reference mapping was established to discriminate between presence or absence of viruses associated with reads. Various criteria for the evaluation of de novo assembled contigs and BLAST results were identified based on virus hits, E-value, percentage query overlap and percentage amplicon overlap. Various RT-PCR systems were used to screen 62 grapevine samples (field collected and candidate nuclear vines) for their virus populations. Seven samples were selected for Illumina MiSeq sequencing, and the data was analysed as per the optimized pipeline. The threshold established for reference mapping and the criteria for BLAST analysis was successfully implemented, proving the applicability of this PCR and HTS-based system in grapevine diagnostics. This system was compared to the standard ELISA system routinely utilised during certification. In our study, when samples evaluated by RT-PCR were tested using ELISA for the presence of GLRaV-1, -2 and -3, a false-negative rate in ELISA of 14.3% was observed, confirming that RT-PCR is the more sensitive test of the two. The capability of RTPCR to readily detect viruses present in low concentrations in woody plants, the availability of primers for virus identification, the ease and rapidity of the technique, together with constant improvement of HTS platforms especially in the area of cost makes this an extremely useful method for grapevine virus diagnostics.
Dissertation (MSc)--University of Pretoria, 2017.
Winetech
Microbiology and Plant Pathology
MSc
Unrestricted

Nous avons pour but de développer un système microfluidique en gouttes, capable, à l’échelle de la cellule/bactérie unique, de détecter et de co-localiser plusieurs marqueurs génétiques, en utilisant une version digitale et multiplexée de la réaction de polymérisation en chaîne (PCR). Les systèmes de PCR digitale actuellement commercialisés ne le permettent toujours pas. Un tel prototype garantira la présence de multiples marqueurs à l’intérieur d’un même génome, ce qui permettra l’identification du pathogène avec précision et un taux de faux-positifs proche de zéro. Comme première application, nous démontrerons la possibilité de co-localiser quatre gènes de virulence de la souche O157:H7 d’Escherichia coli, un pathogène majeur, qui est détecté dans des échantillons alimentaires ou provenant de fèces cliniques pouvant aussi contenir des E. coli non pathogènes porteuses d’une partie des gènes de virulence. Avant de procéder à des tests TaqMan multicolores en point final, E. coli sera d’abord encapsulée dans des gouttes micrométriques et lysée par la chaleur in situ. Notre objectif est de démontrer que ce test peut être appliqué avec succès à un petit ensemble d’échantillons cliniques ou alimentaires
We aim to develop a prototype of droplet-based microfluidic system capable of detecting and colocalizing multiple genetic markers at the single cell/bacteria level, using the Polymerase Chain Reaction (PCR) in a digital multiplexed version. This cannot be achieved using current commercial digital PCR systems, and should increase the sensitivity and reliability of the detection of pathogens. Importantly, the system will guarantee the presence of multiple markers within the same genome and enable accurate identification, and bring the false positive rate close to zero. As a first application, we will demonstrate the possibility to co-localize 3 virulence genes in the E.coli strain O157:H7, a major foodborne pathogen, which has to be detected in clinical feces samples or food samples, which may also contain non pathogenic E. coli carrying only a subset of these virulence genes. E. Coli will be encapsulated in micrometric droplets, lysed by heating in situ prior performing a multicolour end-point Taqman assay. Our objective is to demonstrate that this test can be successfully applied to real clinical or food samples

Background Tuberculosis (TB) infection is a contagious disease due to infection by Mycobacterium tuberculosis(MTB) causing global health burden. There is increasing effort to develop early case detection methods and also to address the issue of multidrug resistance TB (MDR-TB). Molecular methods have been applied to provide rapid and accurate diagnosis. In addition to commercial kits being available for the detection of MTB from clinical specimens, In-house PCR assays have also been developed for the detection of MTB, and can be adjusted according to the laboratories’ own demand. Several molecular techniques like TaqMan probes and Hybridization probes may be applied to target for markers of MTB, e.g. 16s rRNA and IS6110.Detection of the mutation genes, for example, katGfor isoniazid (INH), enables determination of susceptibility of the antibiotic more rapidly than traditional culture methods, and is especially useful due to the increasing emergence of MDR-TB. A wide range of genes have been reported to be related to the resistance of INH, katG315 mutation is the most common gene among them. Therefore, genotyping katG315 allows determination of the susceptibility of INH. Objective The first objective is to evaluate the diagnostic performance of IS6110 One-tube Nested Real-Time PCR for the detection of MTB. Clinical pulmonary specimens collected from Tuen Mun Hospital were retrieved for investigation. All the specimens have already been tested for COBAS TaqMan MTB test and culture results have been obtained for all the samples. During the first stage of the study, all the specimens were tested with IS6110 One-tube Nested Real-Time PCR. Sensitivity, specificity, positive predictive value, negative predictive value and diagnostic odds ratio were obtained from the comparison with the gold standard of MTB detection, i.e., culture. During the second stage of the study, samples were selected to undergo katG315 HybProbe Real-Time PCR assay to determine the genotype of katG. The performance of the assay was evaluated statistically. Result In the first stage of the study, 200 samples were tested with IS6110 One-tube Nested Real-Time PCR. The assay was found to have a sensitivity of 76.92%, specificity of 98.52%, positive predictive value of 96.15%, negative predictive value of 89.86% and the diagnostic odds ratio of 221.667. In the second stage of the study, 66 samples were selected and tested for katG315 HybProbe Real-Time PCR assay, 36 samples were successfully genotyped while 30 samples failed to be genotyped. The only culture proven INH resistance specimen was not amplified at first, and culture isolate was extracted for genotyping again. The repeated test confirmed the genotype of the resistance strain to be a mutant. Conclusion katG315 HybProbe Real-Time PCR assay is a valid approach for genotyping katG. However, the sensitivity and efficiency has to be improved before application for clinical use. From the statistics obtained, COBAS TaqMan PCR assay, which is routinely used in Tuen Mun Hospital, is statistically proven to have comparatively better performance than the IS6110 One-tube Nested Real-Time PCR. Improvement on the assay is required for IS6110 One-tube Nested Real-Time PCR. However, there is great potential of applying both IS6110 One-tube Nested Real-Time PCR and katG315 HybProbe Real-Time PCR assay in clinical use with the same platform available.
published_or_final_version
Microbiology
Master
Master of Medical Sciences

Lung cancer is the leading cause of cancer-related death and is usually diagnosed at advanced stage leading to poor patient survival. Therefore there is a pressing need for early detection of disease. DNA methylation is an early event in carcinogenesis and a limited number of diagnostic markers have been developed for clinical use. This thesis seeks to address whether the development and application of novel DNA methylation assays can diagnose lung cancer at early stage. Previously identified DNA methylation biomarkers, along with novel targets identified by methylation microarray, were developed in multiplex assay format. Twelve markers were used to screen 417 bronchoalveolar lavage specimens from Liverpool Lung Project (LLP) subjects divided into training and validation sets. The optimal biomarker panel (CDKN2A, RARB and TERT) demonstrated improved clinical sensitivity and specificity (Sensitivity/Specificity: 85.7%/93.8%, AUC: 0.91) compared to previous studies. The optimal methylation algorithm detected more than 60% of stage T1 tumours and 93 cytologically occult lung cancer cases. Eight methylated DNA assays were optimised for use with the newly developed Droplet DigitalTM PCR (ddPCR) platform and a targeted pre-amplification technique, MethPlex enrichment, was developed. I established a comprehensive analytical framework to compare performance of methylation-specific ddPCR and quantitative methylation-specific PCR directly and in combination with MethPlex enrichment. ddPCR demonstrated greater precision and linearity, lower limit of detection (WT1 MethPlex ddPCR LOD95 = 1.86 GE), and discriminated twofold differences in methylated DNA input. MethPlex ddPCR detected DNA methylation more frequently in lung cancer patient plasma than in controls in a retrospective case-control study. Technical methylation controls were consistently and precisely detected at inputs as low as 3 methylated copies. Discriminatory efficiency of marker combinations was inadequate, presumably due to limitations in DNA extraction methodology. DNA methylation biomarker diagnostic performance in bronchoalveolar lavage merits further validation in a prospective trial. MethPlex ddPCR analysis showed great promise, demonstrating highly sensitive DNA methylation detection in technical assessment. It is expected that appropriate DNA extraction procedures and higher cfDNA yields will lead to much improved clinical discriminatory efficiency.

Aspergillus fumigatus is a foodborne and airborne human pathogen which produces mycotoxins such as gliotoxin, helvolic acid, fumigallin, and fumigaclavin. The most common disease caused by A. fumigatus is aspergillosis, which is often fatal, especially among AIDS, cancer, and organ-transplant patients. In this study, random cDNA cloning was performed from a cDNA library of A. fumigatus (IMI 385708) constructed on &
#955
ZAP Express. Some of these clones were selected according to their insert sizes and were further subjected to sequence analysis. The sequences were then analyzed by a BLAST search (NCBI Genome Database) to determine the possible functions of these genes. Two of the clones were identified as the primase and 60S ribosomal protein L1-b genes. These genes and a third gene corresponding to the antigenic cell wall galactomannoprotein gene of A. fumigatus were used for the design of three distinct primer pairs. For the primer design, a software was written to differentiate nonconserved regions by multiple sequence alignment. Designed primers were employed in PCR experiments against genomic DNAs of different Aspergillus species. Unique bands were obtained only against A. fumigatus genomic DNA. It was concluded that this PCR-based detection method can be further developed for the rapid detection of A. fumigatus spores from air and food samples.

Background: Currently, there is a lack of non-invasive tumour biomarkers with appropriate sensitivity and specificity to be used in routine clinical testing. The use of circulating microRNAs (miRNAs) as cancer biomarkers has been hypothesised based on their presence and stability in the circulation. Promising initial results for these tiny RNAs has been obtained in the field of breast cancer diagnostics. However, the accurate quantification of circulating miRNAs is more challenging than expected. In particular, several pre- and analytical variables have an impact on their final quantification, including the quantification method. Recently, a new droplet digital PCR (ddPCR) system that can be also used for microRNA quantification has been developed and proved to be very promising in liquid biopsy applications. Experimental design and findings: In order to develop a precise and accurate technique for miRNA quantification, we tested and compared the feasibility of quantifying circulating miRNAs with the new BioRad ddPCR system when used with EvaGreen dye– and TaqMan probe–based assays. In plasma and serum of patients with cancer and healthy controls, two circulating miRNAs and one added exogenous miRNA were quantified with both EvaGreen dye–based miRCURY LNA miRNA assays and TaqMan assays. The EvaGreen-based assay was precise, reproducible and sensitive. In comparison with TaqMan assays, high concordance was obtained for two endogenous miRNAs in serum and plasma. EvaGreen dye–based and TaqMan probe–based assays can be equally used with the ddPCR system to quantify circulating miRNAs. Afterwards, we selected a panel of six miRNAs (miR-10b-5p, miR-145-5p, miR-181a-5p, miR-148b-3p, miR-425-5p, miR-6523p) derived from microarray experiments or described in literature as potential circulating biomarkers for breast cancer. Then, we assessed their absolute levels in two independent cohorts of breast cancer patients and disease-free controls (Italy; n = 56, and USA; n = 94) using EvaGreen-based ddPCR. MiR-148b-3p, miR-181a-5p and miR-652-3p levels were significantly lower in the serum of breast cancer patients than in controls in both cohorts. For these three miRNAs, the stratification of breast cancer patients versus controls was confirmed by receiver operating characteristic curve analysis. Higher levels of serum miR-10b-5p were associated with clinico-pathological features of poor prognosis. These results confirmed the significant discrimination between breast cancer patients and healthy controls and the direction of down regulation. Conclusion: This study establishes the basis for using on a ddPCR for quantifying circulating miRNA biomarkers. Both study cohorts revealed very good agreement in terms of comparable absolute miRNA concentrations and consistent trends of dysregulation in breast cancer patients. This study finally powers the use of circulating miRNAs as cancer biomarkers and proposes miR181a-5p and miR-652-3p as diagnostic biomarker and miR10b-5p as prognostic biomarkers of breast cancer.
Stato dell’arte: L’assenza di marcatori tumorali non invasivi e con appropriata sensibilità e specificità per un uso clinico, rappresenta un problema fondamentale in ambito oncologico. L'utilizzo di microRNA (miRNA) circolanti come biomarker tumorali è stata ipotizzata sulla base della loro presenza e stabilità nel sangue e in altri fluidi biologici. Promettenti risultati preliminari sono stati ottenuti dall’utilizzo di questi piccoli RNA come biomarcatori del cancro al seno. Tuttavia è risultato fin da subito evidente che la quantificazione accurata dei miRNA circolanti è un processo molto complesso e influenzato da molteplici fattori. In particolare non esiste un accordo su quale sia il metodo di quantificazione migliore. E’ stato recentemente sviluppato un nuovo sistema di quantificazione di acidi nucleici chiamato Droplet Digital PCR (ddPCR), ma non era ancora stato testato per la quantificazione di miRNA circolanti. Disegno sperimentale e risultati: Al fine di sviluppare una tecnica precisa ed accurata per la quantificazione dei miRNA circolanti, abbiamo testato l’affidabilità del nuovo sistema ddPCR di BioRad (QX200) e confrontato i risultati della quantificazione dei miRNA circolanti con due chimiche diverse, una basata sull’intercalante EvaGreen e una basata su sonde TaqMan. Nel plasma e siero dei pazienti con cancro e controlli sani, due miRNA circolanti e un miRNA aggiunto esogenamente sono stati quantificati con saggi per miRNA basati su primer a LNA (Exiqon) combinati con EvaGreen o su sonde TaqMan (Applied Biosystem). Entrambi i saggi si sono dimostrati precisi, riproducibili e sensibili. La concordanza tra i dati di quantificazione ottenuti con le due metodiche è risultata essere molto buona. Abbiamo pertanto concluso che sia saggi basati su EvaGreen che sull’uso di sonde TaqMan possono essere ugualmente utilizzati con il sistema ddPCR per quantificare i miRNA circolanti. In seguito, abbiamo selezionato un gruppo di sei miRNA (miR-10b-5p, miR-145-5p, miR-181a-5p, miR-148b-3p, miR-425-5p, miR-652-3p) derivati da esperimenti microarray o descritti in letteratura come potenziali biomarker circolanti per il cancro al seno. Abbiamo quindi valutato i loro livelli assoluti (copie/µl) nel siero in due coorti indipendenti di pazienti con carcinoma mammario e controlli sani (Italia; n = 56, and USA; n = 94) utilizzando saggi ddPCR basati su primer a LNA e sistema EvaGreen. MiR-148b-3p, miR-181a-5p e miR-652-3p sono risultati significativamente più bassi nel siero dei pazienti con cancro al seno rispetto ai controlli in entrambe le coorti. Per questi tre miRNA la stratificazione dei pazienti con carcinoma mammario rispetto ai controlli è stata confermata tramite l’analisi di curve ROC. Inoltre, livelli sierici più elevati di miR-10b-5p sono stati associati con alcune caratteristiche clinico-biologiche a prognosi negativa degli stessi campioni. Conclusione: Questo studio stabilisce la base per l'utilizzo di test basati su ddPCR per la quantificazione di miRNA circolanti quali biomarcatori di tumore al seno. Entrambe le coorti studiate hanno rivelato un ottimo accordo in termini di concentrazioni assolute miRNA e tendenze coerenti di disregolazione in pazienti con cancro al seno rispetto ai controlli. Questo studio suggerisce pertanto l'uso di miRNA come biomarcatori tumorali circolanti e propone miR-181a-5p e miR-652-3p come biomarcatori diagnostici e miR10b-5p come biomarcatore prognostico del tumore al seno.

La viticoltura si trova ad affrontare il problema dalla lotta contro i patogeni che comporta costi ingenti dovuti anche alla sostituzione di piante infette. Le patologie più gravi sono l’Infezione degenerativa, l’Accartocciamento fogliare e il Complesso del legno riccio, tutti causati da infezioni virali. L’impresa cilena Concha y Toro ha deciso di studiare la presenza dei virus all’interno dei propri vigneti in modo da evitarne la diffusione, applicando la tecnica analitica della Real Time PCR, strategia SYBR® Green e TaqMan®. L’obiettivo di questa ricerca è di mettere a punto un protocollo che permetta di rilevare la presenza dei virus applicando una strategia multiplex, al fine di ridurre i costi e i tempi di analisi. Inoltre è possibile inserire un controllo interno che assicuri la veridicità di una risposta negativa alla presenza del virus. Sono state costruite curve standard per le analisi in singleplex dei virus testati dall’azienda e del gene actina di vite, allo scopo di ottimizzare l’analisi. È seguito lo studio delle analisi in duplex prima con l’introduzione del controllo interno e in seguito con test per analizzare combinazioni di virus. Sono state confrontate le efficienze delle curve standard verificando che il valore rimanesse all’interno di un range stabilito e che non ci fosse differenza significativa tra l’analisi in singleplex e la corrispondente in duplex. Lo stesso è stato ripetuto per un protocollo in triplex. Sono state studiate la sensibilità e la specificità di ogni analisi in modo da validare il metodo. I risultati ottenuti hanno dato la possibilità di ottimizzare i protocolli singleplex e confermato la possibilità di introdurre il gene actina come controllo interno nelle analisi. Sono stati raggiunti ottimi risultati anche in molte delle combinazioni in duplex tra virus. In futuro sarà necessario approfondire lo studio delle sensibilità e specificità d’analisi in modo da validare il metodo evitando falsi negativi o falsi positivi.

The aim of this work is to establish novel strategies for the highly sensitive screening of cancer biomarkers in biological samples.To achieve this goal, we developed droplet-based microfluidic dPCR technique. Using a limiting dilution, individual DNA molecules are encapsulated within monodisperse droplets of a water-in-oil emulsion created with a microfluidic device. Fluorescent TaqMan® probes targeting the screened cancer biomarkers allow the detection of mutations. We focused on the mutations in the human KRAS gene for the development of our test. This method is also transposable in a multiplexed format for the parallel detection of multiple mutations in clinical samples.The developed technique allowed the precise quantification of a mutated KRAS gene in the presence of a 200,000-fold excess of un-mutated KRAS genes and enabled the determination of mutant allelic specific imbalance (MASI) in several cancer cell-lines. We validated our technique by screening for KRAS mutations in the blood plasma and tumor samples from patients with metastatic colorectal cancer.

Thesis (MPath)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: Major Depressive Disorder (MDD) is a severe debilitating medical condition that may lead to suicide. Due to a poor understanding of the biological mechanisms underlying the disease process therapeutic decisions are usually taken using a ‘trial and error’ approach. This is not ideal since many treatments do not work as expected for all individuals. Studies have shown that only half of MDD patients receive the appropriate treatment, whereas many patients have adverse response to anti-depressants. These may include weight gain and raised homocysteine levels that may further compromise the health status of MDD patients and may partly explain the link with cardiovascular disease. The objective of the study was to identify genetic risk factors interacting with environmental factors implicated in MDD that may be of relevance to the South African population. Polymorphisms in the MTHFR (677 C>T, rs1801133 and 1298 A>C, rs1801131), COMT (472G>A, rs4680), CYP2D6 (6937G>A, rs3892097), ASMT (24436 G>A, rs4446909) and SLC6A4 (43 bp ins/del, rs4795541) genes were genotyped in 86 MDD patients and 97 population-matched controls. The specific aims were 1) to analytically validate high throughput real-time polymerase chain reaction (RT-PCR) genotyping assays for the selected SNPs against direct sequencing as the gold standard for 2) possible integration into a pathology-supported genetic testing strategy aimed at improved clinical management of MDD. A total of 183 unrelated Caucasians participated in the study, including 69 females and 17 males with MDD and 57 female and 40 male controls without a personal and family medical history of overlapping stress/anxiety and depressive disorders. All study participants were genotyped for the six selected SNPs considered clinically useful based on international data. The allelic distribution of the SNPs, single or combined into a genotype risk score after counting their minor alleles, did not differ between MDD patients and controls. Homocysteine levels were determined and correlated with body mass index (BMI) and other variables known to influence these phenotypes. The folate score assessed with use of the study questionnaire was significantly lower in the patient group compared with controls (p=0.003) and correlated significantly with BMI, particularly in females (p=0.009). BMI was on average 8% higher in the MDD patients compared with controls (p=0.015) after adjustment for age and sex. The MTHFR rs1801133 677 T-allele was associated with a 14% increase in BMI in MDD patients but not controls (p=0.032), which in turn was associated with significantly increased homocysteine levels (p<0.05). The aims of the study were successfully achieved. Identification of the MTHFR rs1801133 677 T-allele reinforces the importance of adequate folate intake in the diet due to increased risk of obesity and depression found to be associated with low dietary intake. Evidence of shared genetic vulnerability for many chronic diseases and drug response mediated by the MTHFR 677 T-allele support the clinical relevance of this low-penetrance mutation.
AFRIKAANSE OPSOMMING: Major depressie (MD) is ‘n aftakelende siektetoestand wat tot selfdood kan lei. Onkunde oor die siekte se onderliggende biologiese meganismes lei dikwels tot ‘n lukrake terapeutiese benadering. Dit is ‘n onbevredigende situasie aangesien indiwidue verskillend reageer op die middels wat voorgeskryf word. Navorsing toon dat slegs ongeveer die helfte van MD pasiënte toepaslike behandeling kry, terwyl anti-depressante ‘n nadelige uitwerking het op baie pasiënte. Dit sluit massatoename en verhoogde homosisteïenvlakke in wat MD pasiënte se gesondheid bykomend nadelig kan beïnvloed en die verband met kardiovaskulêre siekte gedeeltelik kan verklaar. Hierdie studie poog om MD verwante genetiese risikofaktore en omgewingsfaktore wat mekaar beïnvloed en moontlik op die Suid Afrikaanse bevolking betrekking het, te identifiseer. Polimorfismes in die MTHFR (677 C>T, rs1801133 en 1298 A>C, rs1801131), COMT (472G>A, rs4680), CYP2D6 (6937G>A, rs3892097), ASMT (24436 G>A, rs4446909) en SLC6A4 (43 bp ins/del, rs4795541) gene is geanaliseer in 86 MD pasiënte en 97 kontroles geselekteer van dieselfde populasie. Die spesifieke doelwitte was om 1) hoë deurset direkte polimerase kettingreaksie (RT-PCR) genotiperingstoetse vir die 6 gekose polimorfismes met direkte volgordebepaling as maatstaf analities te valideer vir 2) moontlike insluiting in ‘n patologie-ondersteunde genetiese toetsstrategie met die oog op beter kliniese hantering van MD. Altesaam 183 Kaukasiërs het aan die studie deelgeneem. Die MD pasiënte het uit 69 vroue en 17 mans bestaan. Die kontroles (57 vroue en 40 mans) het geen mediese geskiedenis (persoonlik of familie) van oorvleuelende stress/angstigheid of depressie gehad nie. Gebaseer op internasionale data, is al die deelnemers vir die 6 gekose, potensieel klinies-bruikbare polimorfismes getoets. Die alleliese verspreiding van die polimorfismes enkel of gekombineer (uitgedruk as ‘n genotipe-risiko-syfer nadat minor allele getel is), was dieselfde in MD-pasiënte en kontroles. Homosisteïenvlakke is bepaal en gekorreleer met die liggaamsmassa-indeks (BMI) en ander veranderlikes wat bekend is vir hulle invloed op hierdie fenotipes. In teenstelling met die kontroles, was die folaat telling, soos bepaal met die studievraelys, betekenisvol laer in die pasiënte (p=0.003). Die korrelasie met die liggaamsmassa-indeks, spesifiek by vroue, was ook betekenisvol (p=0.009). Na aanpassings vir ouderdom en geslag, is gevind dat die liggaamsmassa-indeks gemiddeld 8% hoër was in die die MD pasiënte teenoor die kontroles. By MD-pasiënte, maar nie by die kontroles nie, is die MTHFR rs1801133 677 T-alleel geassosieer met ‘n 14% toename in liggaamsmassa-indeks (p=0.032), wat ook geassosieer was met betekenisvolle verhoogde homosisteïenvlakke (p<0.05). Die doelwitte van die studie is bereik. Identifisering van die MTHFR rs1801133 677 T-alleel beklemtoon hoe belangrik dit is om voldoende folaat in te neem, veral omdat ‘n verhoogde risiko vir vetsug en depressie met ‘n lae folaatinname in die diet geassosieer word. Die kliniese belang van die MTHFR 677 T-alleel word beklemtoon deur toenemende bewyse wat daarop dui dat gedeelde genetiese vatbaarheid vir ‘n verskeidenheid van kroniese siektes asook middelrespons aan bemiddeling deur hierdie lae penetrasie mutasie toegeskryf kan word.
Winetech
Technology for Human Resources and Industry Program (THRIP).

碩士
國立嘉義大學
獸醫學系研究所
99
P-glycoprotein is the translation protein of the multidrug resistant gene (MDR1). Its activity affects the sensitivity of drugs in animals directly. Multidrug resistant genes may affect the functions of P-glycoprotein through heredity mutations. This effect causes drugs to pass the blood-brain barrier and accumulates in the brain, which harms the nervous system; this may even lead to death. Recent methods of diagnosing P-glycoprotein heredity mutations (mdr1-1Δ mutation) are time-consuming. This research is about rapidly screening mdr1-1Δ mutations based on PCR diagnosing techniques and the screening rate of canines in Taiwan using this method. This experiment uses self-designed primers to process multiplex PCR. The results successfully distinguish canines with normal genes and canines with complete/single defected genes. The tests were run on 265 canines, which include 11 different species. The results indicated 5 canines have single defected genes. Two of them were Shetland Sheepdogs (2/13) and three of them were Old English Sheepdogs (3/23).The rapid diagnosing method of the mdr1-1Δ gene mutation used in this research may benefit in evaluating drug uses in animal clinical practice.

The samples under investigation were collected in Department of Pediatric Infectious Diseases of the University Hospital (Brno). Group of patients (100) was heterogeneous in terms of symptoms, age and sex. The samples were taken from patients with an LB diagnosis and from those with nonspecific symptoms. Molecular typing of LB spirochetes in clinical samples (104 blood/serum, 89 cerebro-spinal fluid and 1 synovial fluid) became necessary when the general immunological tests gave unclear results. The samples were analyzed using PCR-based and molecular biology techniques that include: nested- and spacer-PCR, specie-specific PCR, sequence, virtual hybridization, in silico RFLP analysis, similarity search. Results of conducted analysis confirmed that 51% of samples (98) were positive on B. bugrdorferi sensu lato. Using above mentioned techniques 6 spirochete species from B. burgdorferi sensu lato complex were identified; two of them weren?t detected in samples of human origin in Europe yet. Comparative analysis of two media for Borrelia cultivation from samples of human origin definitely proved the adventage of using MKP instead of traditionally used BSK-H Complete.

碩士
高雄醫學大學
臨床醫學研究所
105
Background The culture method has been strongly emphasized in the clinical setting of Helicobacter pylori (H. pylori) eradication failure because retreatment strategy can be tailored based on the culture antibiotics susceptibility test. However, the culture method yields low accuracy, especially in treatment-experienced H. pylori infection. The current clinical unmet need in the setting of treatment-experienced H. pylori infection is one diagnostic method with satisfying accuracy as well as the ability to perform the antibiotics susceptibility test. Theoretically, gastric juiced-based PCR is able to provide both strengths, but there is no clinical data. Our study intended to provide the data regarding the diagnostic accuracy of gastric juice-based PCR in the treatment-experienced H. pylori infection and compare the results of gastric juice-based PCR with that of the culture method. Methods We included 711 patients and categorized them into 4 groups based on their previous treatment history: treatment-naïve, post 1st line therapy, post 2nd line therapy and post 3rd line therapy. The status of H. pylori infection in each subject was confirmed according to the following clinical gold standards: concordant positive histology and rapid urease test or positive urease breath test. We performed gastric juice-based PCR and culture in each subject and then calculated the sensitivity, specificity, positive predictive value, negative predictive value and accuracy of gastric juice-based PCR as well as culture method. Finally, we compared the results with Fisher’s exact test. Results Our findings demonstrated that the accuracy of gastric juice-based PCR was higher than that of the traditional culture method in treatment-naïve patients (96% vs 87%, p values = 0.0001), in patients post 1st line therapy (97% vs 79%, p values = 0.0002), in patients post 2nd line therapy (96% vs 77%, p values = 0.0012) and in patients post 3rd line therapy (100% vs 70%, p values = 0.0092). The sensitivity of gastric juice-based PCR was also better than that of the traditional culture method in treatment-naïve patients (92% vs 58%, p values < 0.0001), in patients post 1st line therapy (90% vs 35%, p values < 0.0001), in patients post 2nd line therapy (93% vs 60%, p values = 0.0006) and in patients post 3rd line therapy (100% vs 22%, p values = 0.0023). The specificity, positive predictive and negative predictive values were calculated as well. The results indicated that gastric juice-based PCR outperformed the traditional culture method in diagnosis of treatment-experienced H pylori. Conclusion The gastric juice-based PCR yields more accurate diagnosis in treatment-experienced H. pylori than the traditional culture method and also provides the anti-microbial susceptibility test to guide subsequent retreatment therapy. Our study demonstrated that gastric juice-based PCR suits the current unmet need for the management of treatment-experienced H pylori and has very promising potential for widespread application in the setting of treatment failure.