Dissertations / Theses on the topic 'Real-time PCR'
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Crane, Bryan Lee 1976. "Real time PCR measurement by fluorescence anisotropy." Thesis, Massachusetts Institute of Technology, 2004. http://hdl.handle.net/1721.1/30347.
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Includes bibliographical references (p. 181-190).
Real-time polymerase chain reaction (PCR) is the gold-standard for quantitation in both mutation and gene expression analyses. Already this technique has found valuable clinical application in disease diagnosis and progression evaluation. As the number of known gene-disease correlations continues to rise, there will be increased demand for higher throughput and decreased cost for these analyses. Present real-time PCR measurement is based upon the fluorescent intensity of either intercalating dyes or oligonucleotide probes. Intercalating dye methods suffer from a lack of binding specificity, while probe methods are expensive and require increased assay optimization. In this thesis, a new method is presented for monitoring real-time PCR that utilizes the fluorescent anisotropy (FA) of labeled primers. FA, when measured at constant temperature, is indicative of the molecular mass to which the fluorophore is attached. Specificity is improved with the FA method over the use of intercalating dyes since the selective binding of primers is required for signal change. Assay complexity and cost are reduced compared to fluorogenic probe methods since the probes are eliminated. The design of a prototype instrument, which successfully implements this new method, is presented. Instrument and assay performance are compared to intercalating dye assays run in commercially available instrumentation. Theoretical limits on performance are also presented and compared to experimental results. Excellent repeatability and linearity are observed with respect to these benchmarks. This new method, having both high specificity and low optimization complexity, is expected to be particularly applicable to the demanding robustness requirements of nano-scale PCR.
by Bryan Lee Crane.
Ph.D.
Rozales, Franciéli Pedrotti. "Real time-pcr e nested-pcr no diagnóstico da tuberculose pulmonar." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2013. http://hdl.handle.net/10183/72990.
Full textTuberculosis (TB) remains as an important public health problem worldwide. Therefore, the rapid detection of M. tuberculosis is of primary importance to effectively reduce transmission among patients. The aims of this study were to evaluate two molecular tests to detect M. tuberculosis complex (MTBC) directly from clinical samples. The study included 124 respiratory samples which were evaluated by two in house molecular assays for MTBC detection: Nested PCR (NPCR) and Real Time PCR (RT-PCR). The respiratory samples were also evaluated by the direct test (AFB assay). The results were compared with the results of culture and also compared with the culture results plus clinical data of patients. We used a commercial DNA sample with known quantification to establish the Limit of Detection (LOD). The LOD was 1 copy/μL for RT-PCR and 25 copies/μL for NPCR. The AFB assay presented low sensitivity – SE - (40%) and a high specificity - SP – (94%). Both molecular assays, RT-PCR and NPCR presented high SE and SP (RT-PCR 98% and 91%, NPCR 86% and 93%, respectively) compared to culture. When the results of the molecular tests were compared to the culture plus clinical data the SE and SP were 90,20% and 97,26% for RT-PCR and 80,39% and 98,63% for the NPCR, respectively. It was possible to observe a slight decrease of SE of the molecular methods in comparison to culture plus clinical data in relation to culture; however, the SP was increased, since many cases of TB could not be confirmed by culture. Furthermore we evaluated the cost of molecular assays: the NPCR cost was $17.77/test while the RT-PCR cost was $15.76/test. The RT-PCR test was faster (2 hours) than the NPCR (4 hours) to be performed. Our study confirms that PCRs may be useful for rapid diagnosis of respiratory TB, with high SP rates. It may also be very important to exclude such diagnosis, considering the high NPV found in our study. In summary, PCRs targeting IS6110 of MTB improve the accuracy of the diagnosis of pulmonary TB, with many potential positive effects for clinical management and control of the disease.
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.
Full textApontada 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.
Dunkley, Kingsley Delroy. "Modulation of cell yields and genetic responses of Salmonella fermentation and colonization in the gastrointestinal ecology of avian species." [College Station, Tex. : Texas A&M University, 2006. http://hdl.handle.net/1969.1/ETD-TAMU-1187.
Full textAndalo, Alice. "Analisi quantitativa dell'espressione genica mediante real-time rt-pcr." Bachelor's thesis, Alma Mater Studiorum - Università di Bologna, 2015. http://amslaurea.unibo.it/8450/.
Full textDörries, Hans-Henno. "Entwicklung von Real-Time-PCR-Nachweissystemen für getränkerelevante Hefen." [S.l.] : [s.n.], 2006. http://deposit.ddb.de/cgi-bin/dokserv?idn=979663938.
Full textHartmann, Britta. "Entwicklung einer Real-time-PCR-Nachweismethode für Yersinia enterocolitica." [S.l.] : [s.n.], 2007. http://edoc.ub.uni-muenchen.de/archive/00006660.
Full textHartmann, Britta. "Entwicklung einer Real-Time PCR-Nachweismethode für Yersinia enterocolitica." Diss., lmu, 2007. http://nbn-resolving.de/urn:nbn:de:bvb:19-66603.
Full textMalatji, Dikeledi Petunia. "Detection of Babesia rossi genotypes using real-time PCR." Diss., University of Pretoria, 2011. http://hdl.handle.net/2263/31138.
Full textDissertation (MSc)--University of Pretoria, 2011.
Veterinary Tropical Diseases
MSc
Unrestricted
Zhang, Yan. "Frequent RASSF1A gene promoter hypermethylation in breast cancer." [S.l. : s.n.], 2008. http://nbn-resolving.de/urn:nbn:de:bsz:289-vts-63611.
Full textLandgraf, Maria. "Detection of food relevant filamentous fungi by real time PCR." [S.l.] : [s.n.], 2006. http://deposit.ddb.de/cgi-bin/dokserv?idn=98023946X.
Full textTolardo, Aline Lavado. "Desenvolvimento e aplicação de RT-PCR em tempo real para Vesiculovirus brasileiros." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/17/17138/tde-21102015-110512/.
Full textThe Vesiculovirus is a Rhabdoviridae family genre of RNA virus that includes serotypes Carajás, Cocal, Maraba, Piry, Alagoas and Indiana. These are causes of vesicular stomatitis in ruminants and human febrile illness in Brazil. The vesiculoviroses and its epidemiology are little known in humans. Still, Vesiculovirus (VSV) are poorly diagnosed in humans and laboratory animals by the lack of diagnostic methods. Therefore, we proposed in this work to develop and test a real-time RT-PCR by SYBR Green method, focusing on the detection of Brazilian VSV. Primers that amplify part of the VSV G protein gene were used in the test which proved capable of detecting genomes of VSV Piry, Indiana, Alagoas and Carajás. The method was used to test serum samples from patients with acute febrile disease, cattle, horses and macerated arthropods. Real time RT-PCR showed to be 100 times more sensitive than conventional RT-PCR for Vesiculovirus and also was possible to detect up to 10 RNA copies of the Piry virus. Also, the real-time RT-PCR for Vesiculovirus proved able to diagnose and quantify Alagoas VSV in serum samples from cattle and horses. Therefore, the real-time RT-PCR developed in this work will probably be very useful in the diagnosis and in future research, which will increase the epidemiological knowledge, as it is still little known about the Vesiculovirus.
Steyn, HC, A. Pretorius, and CME McCrindle. "A quantitative real-time PCR assay for Ehrlichia ruminantium using pCS20." Elsevier, 2008. http://encore.tut.ac.za/iii/cpro/DigitalItemViewPage.external?sp=1000379.
Full textDib, Cristina Corsi. "Emprego da reação em cadeia pela polimerase em tempo real para o controle de eficiência de bacterinas anti-leptospirose." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-27092012-164459/.
Full textLeptospira interrogans serovar Kennewicki strain Fromm was used for the production of a experimental leptospirosis bacterin. The extraction of total RNA used for reverse transcription and quantification of the antigens LigA and LipL32 for Real Time PCR was performed from the aliquots harvested of bacterin dilutions before inactivation that were separated and maintained at -80ºC. The remaining volume of bacterin was inativated at 56ºC and maintained at -20ºC for the evaluation bacterin potency in hamsters and detection and quantification of LigA and LipL32 antigens by Indirect ELISA assay and Indirect Sandwich ELISA. The results of potency assay in hamsters demonstrated that the bacterin was approved by the international patterns of quality until dilution 1/6400, protecting the hamters against lethal infection challenge by the dilution 10-6 (100 infectious doses 50%/0,2 mL). The results of Real Time PCR detected 3,2 x 103 e 2,3 x 101 copies of mRNA that encodes the LigA protein, in samples of pure bacterin and diluted 1:200, respectively. Few eight copies of mRNA that encodes LipL32 protein were detected in pure bacterin samples. Indirect ELISA assays not detected LigA protein in inactivated bacterin samples, but demonstrated LipL32 protein detection until dilution 1:1600 of bacterin. Indirect Sandwich ELISA presented cross-reaction in control plates, so the results cannot be considerated in the analysis. The results of real time PCR cannot be correlated with the potency assay in hamsters but Indirect ELISA assay for protein LipL32 demonstrated that the results were suitable with the results presented by the potency assay in hamsters offering a possible in vitro alternative for the evaluation of leptospirosis bacterins potency.
Pantchev, Alexandra. "Spezies-spezifischer Nachweis von Chlamydien bei Haustieren mittels Real-Time PCR." Giessen VVB Laufersweiler, 2010. http://geb.uni-giessen.de/geb/volltexte/2010/7676/index.html.
Full textWilhelm, Jochen. "Entwicklung real-time-PCR-basierter Methoden für die moderne DNA-Analytik." [S.l. : s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=96787775X.
Full textTichopád, Aleš. "Quantitative real-time RT-PCR based transcriptomics improvement of evaluation methods /." [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=972765069.
Full textAfshari, Kashanian Elisa. "Detection of celery (Apium graveolens) in food with Real-Time PCR." Thesis, Uppsala University, Department of Medical Biochemistry and Microbiology, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7130.
Full textDirective EC 2003/89/EC of the European Parliament and of the Council states that certain
ingredients and products derived there of known to cause allergen reactions must always be
declared. Furthermore labelling is mandatory irrespective of the amount included. The National
Food Administration therefore needs methods for monitoring the presence of allergens in food.
Methods already exist for most of the allergens on the EU-list, but an operational method for
celery (Apium graveolens) is missing.
A specific DNA-method was developed, based on TaqMan Real-Time PCR with the celery
mannitol dehydrogenase gene as target sequence. The analysis was started with homogenisation
of the sample followed by extraction of DNA. The Real-Time PCR method was shown to be
specific for celery, producing a 113 bp fragment with two celery varieties and negative results
with other closely selected species commonly present together with celery in food products (12
samples). The detection limit was 2-20 pg DNA, which corresponds to 1-7 haploid genome
copies. When evaluated with model samples of celery in meat, a detection limit of less than
0,01 % was determined. When used to analyse food products from the market, six out of seven
products declared to contain celery were correctly identified as positive.
Ikuta, Koji, Toshio Sasao, Yuya Okuda, Kenji Watamura, and Masashi Ikeuchi. "Development of New Biochemical IC Chip-Set for Real-Time PCR." IEEE, 2009. http://hdl.handle.net/2237/13926.
Full textBenninghoff, Myrna. "Etablierung und Evaluierung eines molekularen Wurmdiagnostikverfahrens (Real-Time-PCR) in Tansania." Diss., Ludwig-Maximilians-Universität München, 2015. http://nbn-resolving.de/urn:nbn:de:bvb:19-182939.
Full textKelley, Erin, Elizabeth Driebe, Kizee Etienne, Mary Brandt, James Schupp, John Gillece, Jesse Trujillo, et al. "Real-time PCR assays for genotyping of Cryptococcus gattii in North America." BioMed Central, 2014. http://hdl.handle.net/10150/610059.
Full textHipólito, Janayna Roriz. "Diagnóstico molecular para malária por nestedpcr e pcr em tempo real." Universidade Federal do Amazonas, 2006. http://tede.ufam.edu.br/handle/tede/2242.
Full textFundação de Amparo à Pesquisa do Estado do Amazonas
A malária é um problema de saúde pública na região Amazônica, mais de 200 mil casos dessa doença ocorrem anualmente no Amazonas, sendo 80% deles causados pelo P. vivax, que vem apresentando índices crescentes de morbidade, principalmente associados à diminuição da sensibilidade aos antimaláricos. Dentre as estratégias para combate e controle da doença, a identificação rápida e precisa da espécie é ferramenta indispensável para um tratamento apropriado, diminuição do risco de transmissão e melhor entendimento da epidemiologia desses parasitas. A técnica microscópica da gota espessa é a principal para diagnóstico da malária, entretanto, outros métodos vêm sendo testados, principalmente os moleculares que tem se mostrado mais sensíveis e específicos para detectar e diferenciar as espécies em baixas parasitemias. Avanços desse método, como a PCR em tempo real, permitem que o resultado do teste seja detectado simultaneamente a amplificação, diminuindo o tempo gasto para a realização do diagnóstico. Com o intuito de detectar molecularmente a malária, verificando a presença de plasmódios, o diagnóstico molecular foi realizado através das técnicas de PCR em tempo real e nested-PCR, para se fazer uma comparação desses dois métodos com o diagnóstico microscópico da gota espessa em 300 amostras criopreservadas, 200 coletadas no dia inicial do tratamento (D0), das quais 88% (176/200) eram provenientes de pacientes de Manaus, as 24 amostras restantes (12%) eram provenientes de localidades do interior do Amazonas: São Gabriel da Cachoeira (09), Tefé (08), Humaitá (04) e Careiro (03). Apenas 9% dessas amostras tinham diagnóstico microscópico de monoinfecção por P. falciparum e 91% (182/200) por P. vivax, não havia nenhuma amostra mista pelo diagnóstico microscópico. O diagnóstico molecular por nested-PCR confirmou a presença de DNA de plasmódio em 100% das amostras monoinfectadas. Adicionalmente, foram observadas infecções mistas, co-infecção de P. falciparum e P. vivax, em 19% (38/200) destas amostras. O diagnóstico molecular por PCR em tempo real (Lightcycler, Roche®) foi realizado em apenas 17% (34/200) dessas amostras. A co-positividade (sensibilidade) dos testes para P. vivax foi em média 71% e a co-negatividade (especificidade) 92%, para P. falciparum a co-positividade foi 91% e a conegatividade 79%. A concordância entre os testes foi regular. As 100 amostras restantes haviam sido coletadas no sétimo dia (D7) de tratamento e eram negativas pela microscopia. O diagnóstico molecular demonstrou 21% de positividade. Este estudo mostrou que muitas infecções mistas vêm sendo subestimadas para fins de avaliação epidemiológica, demonstrando que a sensibilidade e especificidade do diagnóstico molecular são superiores a do teste microscópico. O diagnóstico molecular seria então mais indicado como teste complementar no diagnóstico de pacientes com baixas parasitemias, na análise da quantidade de portadores assintomáticos, em estudo de infecções criptônicas e na avaliação da negativação da parasitemia para monitoramento terapêutico, e em estudos que visem a diminuição da transmissão pela existência de prováveis gametócitos persistentes após o tratamento. Entretanto, esse método não é indicado para rotina de diagnóstico de malária, uma vez que os resultados positivos por essa técnica não significam necessariamente que o paciente desenvolva a doença.
Elfaitouri, Amal. "Development of Real-Time PCR Based Methods for Detection of Viruses and Virus Antibodies." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7320.
Full textPang, Zhenyi. "Non-alcoholic fatty liver disease : real-time PCR analysis of gene expression /." [St. Lucia, Qld.], 2004. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe.pdf.
Full textAndréasson, Hanna. "Sensitive Forensic DNA Analysis : Application of Pyrosequencing and Real-time PCR Quantification." Doctoral thesis, Uppsala University, Department of Genetics and Pathology, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-5775.
Full textThe field of forensic genetics is growing fast and the development and optimisation of more sensitive, faster and more discriminating forensic DNA analysis methods is highly important. In this thesis, an evaluation of the use of novel DNA technologies and the development of specific applications for use in forensic casework investigations are presented.
In order to maximise the use of valuable limited DNA samples, a fast and user-friendly Real-time PCR quantification assay, of nuclear and mitochondrial DNA copies, was developed. The system is based on the 5’ exonuclease detection assay and was evaluated and successfully used for quantification of a number of different evidence material types commonly found on crime scenes. Furthermore, a system is described that allows both nuclear DNA quantification and sex determination in limited samples, based on intercalation of the SYBR Green dye to double stranded DNA.
To enable highly sensitive DNA analysis, Pyrosequencing of short stretches of mitochondrial DNA was developed. The system covers both control region and coding region variation, thus providing increased discrimination power for mitochondrial DNA analysis. Finally, due to the lack of optimal assays for quantification of mitochondrial DNA mixture, an alternative use of the Pyrosequencing system was developed. This assay allows precise ratio quantification of mitochondrial DNA in samples showing contribution from more than one individual.
In conclusion, the development of optimised forensic DNA analysis methods in this thesis provides several novel quantification assays and increased knowledge of typical DNA amounts in various forensic samples. The new, fast and sensitive mitochondrial DNA Pyrosequencing assay was developed and has the potential for increased discrimination power.
Muradrasoli, Shaman. "Detection and Quantification of Variable Viral RNA by Real-Time PCR Assays." Doctoral thesis, Uppsala universitet, Klinisk virologi, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-9193.
Full textMohamed, Nahla. "Molecular Diagnosis of Common Viral Infectious Diseases Based on Real-Time PCR." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7118.
Full textAndréasson, Hanna. "Sensitive forensic DNA analysis : application of pyrosequencing and real-time PCR quantification /." Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-5775.
Full textChan, Chi-chiu Elvin, and 陳志超. "Molecular characterization of toxigenic clostridium difficile by multiplex and real-time PCR." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B46631392.
Full textLeopold, Luciana Eleanor Dittmer Dirk Peter. "Development of real-time PCR assays for the quantitative detection of herpesviruses." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2008. http://dc.lib.unc.edu/u?/etd,1487.
Full textTitle from electronic title page (viewed Sep. 16, 2008). "... in partial fulfillment of the requirements for the degree of Master of Science in the Curriculum of Genetics and Molecular Biology." Discipline: Genetics and Molecular Biology; Department/School: Medicine.
Ferreira, Karin Correa Scheffer. "Detecção do vírus da raiva em órgãos de morcegos do gênero Artibeus (Leach, 1821) por meio de RT-PCR, Hemi-Nested RT-PCR e Real Time RT-PCR." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-19102012-132944/.
Full textThis study was aimed to detect the presence of rabies virus in different organs of the genus Artibeus bats using molecular techniques such as RT-PCR, hnRT-PCR, and the Real Time RT-PCR. From about 4,000 specimens of bats received for rabies diagnosis at the Pasteur Institute, 30 bats of the genus Artibeus were then selected. The selected bats presented positive results by the traditional DFA and N2A-cells inoculation test using brain tissue suspensions. Samples of salivary glands, urinary bladders, kidneys, lungs, and fecal contents and washings of the skulls were collected for the molecular techniques testing. The organs and the fecal contents were diluted at 1:10 (w/v) and the urinary bladder, at 1:20 (w/v) and these suspensions were inoculated into N2A cells for viral isolation. The extraction of the total RNA was performed by using TRIzol® and followed by the reverse transcription and the PCR and the hnRT-PCR were performed by using specific primers for the gene encoding the protein N. The product obtained by the reverse transcription technique was submitted to the Real Time RT-PCR technique, using primers and probe specific for antigenic variant 3 of the rabies virus. Of the 30 suspensions of the brain washings, 28 (93.33%) were positive in N2A cell culture inoculation, followed by the suspensions of the salivary glands (36.67%), bladders (16.67%) and fecal contents (3.33%). For the 180 samples evaluated, the results of sensitivity found for the RT-PCR, hnRT-PCR and Real Time RT-PCR techniques were 56.25%, 82.57%, and 82.19%, respectively. A comparison of hnRT-PCR and Real Time RT-PCR techniques performed by Fisher\'s exact test showed that the proportion of positives detected by the brain washings, organs and of the fecal content was non-significant (P> 0.05). Regarding the results found in hnRT-PCR and Real Time RT-PCR techniques, 100% positives were in brain washing, 90% and 93.33% in salivary glands, 83.33% and 90% in bladders, 80% and 93.33% in kidneys, 76.67% and 50% in lungs and 43.33% for both techniques on fecal contents. These results suggest that both hnRT-PCR and Real-Time PCR techniques can be used as complementary methods for the diagnosis of rabies and are sensitive enough for use in pathogenesis studies. The Real Time RT-PCR technique performed in this study proved to be faster and more sensitive and effective in detecting RABV in different organs and extra neural tissues of bats.
Nascimento, Cristiane Santos. "Uso de método de biologia molecular quantitativo (PCR real-time) na avaliação da carga parasitária em cães naturalmente infectados por Leishmania sp." s. n, 2011. https://www.arca.fiocruz.br/handle/icict/4152.
Full textMade available in DSpace on 2012-06-25T21:03:51Z (GMT). No. of bitstreams: 1 Cristiane Santos Nascimento Uso de método de biologia molecular....pdf: 1323686 bytes, checksum: caf3acf13d85858d02cc05e45a821e9c (MD5) Previous issue date: 2011
Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil
INTRODUÇÃO: A leishmaniose visceral humana (LVH) é uma importante causa de morbidade e mortalidade no Brasil. Apesar dos avanços no conhecimento da epidemiologia da LVH, ainda existem lacunas importantes nas informações sobre os principais reservatórios desta zoonose. A técnica validada para avaliação da infectividade de reservatórios, xenodiagnóstico, é um método laborioso, demorado e difícil de executar, portanto, inapropriado para a triagem de grande número de animais. A padronização de método capaz de quantificar a carga parasitária presente em diferentes tecidos pode oferecer respostas importantes sobre a epidemiologia e a prevenção da LVH. OBJETIVO: Avaliar a carga parasitária em diferentes amostras biológicas de cães naturalmente infectados por Leishmania chagasi, utilizando método de biologia molecular quantitativo, PCR real-time (qPCR). MÉTODOS:Entre nov/2004 e abr/2007, foram realizados seis inquéritos soro-epidemiológicos em duas áreas endêmicas para LVH. Os cães soropositivos foram eutanasiados e submetidos a: exame de cultura, parasitológico direto e exame histológico para confirmação da infecção. Amostras de sangue periférico e fragmento de pele foram coletadas em todos os animais para determinação da carga parasitária. Adicionalmente, coletou-se também swab da conjuntiva, aspirado de medula óssea e linfonodo para realização do teste de qPCR nos cães incluídos no último inquérito (abr/2007). A técnica de qPCR foi padronizada utilizando um par de primers LEIF e LEIR e sonda LEIP selecionados no gene SSu rRNA. A seleção dos primers e sonda foi realizada utilizando o programa Primer Express (Perkin-Elmer-Applied Biosystems). A sonda fluorogênica foi sintetizada utilizando uma molécula FAM ligada na extremidade 5‟ e TAMRA ligada à extremidade 3‟(Perkin-Elmer -Applied Biosystems). Para determinar a carga parasitária foi realizada curva padrão com o DNA obtido da cultura de L. chagasi em concentrações variando de 101 a 107 parasitas/ml. Cada ponto da curva foi testado em triplicata. RESULTADOS: Dos 98 cães soropositivos identificados, foi detectado DNA de Leishmania em 57% das amostras de sangue total, em 56% das amostras de pele e em 100% das amostras de medula óssea, linfonodos e swab da conjuntiva. A carga parasitária em sangue periférico e swab da conjuntiva não ultrapassou 103 parasitas/ml sendo mais comumente detectado 1 a 10 parasitas/ml. Por outro lado, em pele, medula óssea e linfonodos a carga parasitária passou de 104 parasitas/ml, além disso, as quantidades de DNA detectadas se distribuíram com maior freqüência na categoria acima de 104 parasitas/ml, notadamente em amostras de linfonodos. CONCLUSÕES: O qPCR apresentou alta sensibilidade nas amostras biológicas estudadas, particularmente em linfonodos , medula óssea e pele. Nossos resultados indicam que o qPCR pode ser utilizado numa variedade de amostras biológicas para a quantificação da carga parasitária de cães naturalmente infectados por Leishmania sp. Estudos de validação do qPCR para avaliar a capacidade de reservatórios da LV, em lugar do xenodiagnóstico, e para investigar o papel do qPCR na triagem de cães em programas de controle/prevenção da LV devem ser conduzidos.
INTRODUCTION: Human visceral leishmaniasis (LVH) is an important cause of morbidity and mortality in Brazil. Despite advances in knowledge of the epidemiology of LVH, there are still important gaps in information on the main reservoirs of this zoonotic disease. The validated technique for assessing the infectivity of reservoirs, xenodiagnosis, is laborious, time consuming and difficult to implement, therefore, inappropriate for screening large numbers of animals. A standardized method to quantify the parasite load present in different tissues may provide important answers on the epidemiology and prevention of LVH. OBJECTIVE: To assess the parasite load in different biological samples from dogs naturally infected by Leishmania chagasi using a molecular biology quantitative method, real-time PCR (qPCR). METHODS: From nov/2004 to apr/2007, six seroepidemiological surveys were conducted in two endemic areas for LVH. The seropositive dogs were euthanized and submitted to: culture, direct parasitological and histological examination to confirm infection. Blood samples and skin fragments were collected in all animals to determine the parasite load. Additionally, conjuntival swabs, bone marrow and lymph node aspirates were also collected to do qPCR in dogs included in the last survey (apr/2007). The qPCR technique was standardized using a pair of primers and probe and LEIF /LEIR and LEIP selected in the SSU rRNA gene. The selection of primers and probe was performed using the program Primer Express (Perkin-Elmer-Applied Biosystems). The fluorogenic probe was synthesized using a FAM molecule attached at the 5 'end and TAMRA linked to the 3' end (Perkin-Elmer-Applied Biosystems). In order to determine the parasite load a DNA standard curve was plotted with DNA obtained from L. chagasi culture in concentrations ranging from 101 to 107 parasites/ ml. Each point on the curve was tested in triplicate. RESULTS: Of the 98 seropositive dogs identified Leishmania DNA was detected in 57% of the whole blood samples, 56% of the skin samples and 100% of bone marrow, lymph nodes and conjuntival swabs samples. The parasite load in peripheral blood and conjuntival swab did not exceed 103 parasites/ml, and was more commonly in the range of 1-10 parasites /ml. On the other hand, skin, bone marrow and lymphnode parasite burden exceeded 104 parasites / ml, in addition, the quantities of DNA detected were distributed more frequently in the category above 104 parasites/ml, especially in lymphnodes samples. CONCLUSIONS: qPCR showed high sensitivity in biological samples studied, particularly in lymphnodes, bone marrow and skin. Our results indicate that qPCR could be used in a variety of biological samples to quantify the parasite load in dogs naturally infected by Leishmania sp. qPCR validation studies to assess potential reservoirs for VL (replacing xenodiagnosis), and to investigate the role of qPCR in dog screening programs for the control/prevention of LV should be conducted.
Rosa, Stefanie Ulrike. ""Real-Time-PCR-Untersuchungen zur Persistenz von infektiösen Toxoplasma-gondii-Dauerstadien in Rohwurst-Erzeugnissen"." Giessen VVB Laufersweiler, 2009. http://d-nb.info/996004408/04.
Full textMidena, Raquel Zanin. "Suscetibilidade antimicrobiana e protocolo de purificação de RNA para análise de expressão gênica de isolados clínicos de Fusobacterium nucleatum." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/25/25147/tde-26022016-143429/.
Full textFusobacterium nucleatum is a Gram-negative bacterial species, strict anaerobic and one of the species often found in primary infection of the root canal system. This species has great importance in biofilm formation to be a union bridge between species which are not able to act alone. The constituent microorganisms of the biofilm exchange each tother genetic material, increasing the strength of them. It is almost impossible for a clinical isolate have genes totally equal to a standard strain, such as strains of culture collections like ATCC (American Type Culture Collection). The present study investigated anaerobic bacterial species Fusobacterium nucleatum isolated from root canals, comparing them to the ATCC strain. The microbial in vitro susceptibility of biofilm and planktonic growth of the strains was compared by means of microbiological culture and the E-test method, with the antibiotics Amoxicillin, Amoxicillin + clavulanic acid, Ampicillin, Azithromycin, Clindamycin, Eritromycin and Metronidazole.. Also, a RNA Purification protocol for the strains under the same growth conditions was defined. Clinical isolates were obtained by microbiological samples of patients with teeth with pulp necrosis and apical periodontitis visible on radiographs. The species isolation and identification were performed using commercial biochemical tests (Sistema Api, Bio-Meriéux, France) and conventional PCR, obtaining four clinical isolates. The protocol for RNA purification was done with zirconia beads, bead beater device and commercial kit RNeasy (Qiagen) and transcribed into complementary DNA (cDNA). The quality of purification was tested for its ability of amplification by real time polymerase chain reaction (qPCR) using primer for the gene 16s RNA specific for F. nucleatum. All tested trains were 100% susceptible to amoxicillin + clavulanic acid, ampicillin, azithromycin, clindamycin and metronidazole. Both types of bacterial growth showed resistance to Erythromycin. Bacteria in biofilm showed a decrease in susceptibility to all antibiotics, but without statistical difference. The protocol proposed for the purification of RNA of F. nucleatum was effective, in the planktonic and the biofilm growth. The average yield of RNA samples for bacteria in planktonic growth was 514.2 ng /μL (SD ± 397.7) and the samples in biofilm was 377.1 ng / μL (SD ± 144.1). These found values suggest a good quality of RNA, free of protein contamination. The established protocol for the purification of the RNA of the Fusobacterium nucleatum strains, grown in biofilm and planktonic phase, had successfully amplified by qPCR.
Bernardes, Nara Thiers Cacciatori Galleti. "Desenvolvimento PCR em Tempo-Real em sistema TaqMan® para detecção de rotavírus em amostras clínicas de bovinos." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-26092016-103616/.
Full textNeonatal diarrhea is the main health problem affecting the calves in the first weeks of life, causing major losses due to morbidity, mortality, treatment costs and delayed development. Rotaviruses are the most important causative agents of viral gastroenteritis in children and in different animal species. In addition to its economic impact on livestock, cattle can act as reservoirs for genetic and antigenic diversity for human samples. Therefore, the diagnosis of this agent is critical to the development of more specific preventive measures for their control. The objective of this work is to develop a method of PCR for rotavirus detection in cattle using TaqMan® system targeting the 5 nonstructural protein (NSP5). For this, 113 samples of cattle feces were collected from farms of São Paulo, and previously tested by convencional PCR. To PCR standardization, the standard sample was cloned, generating plasmid 3x1010 copies/reaction. The β-actin was usede as exogenous control. The limit of detection was determined by serial dilutions, to detect 6 x101 copies/µl. The standard curve of PCR to encoding segment detection NSP5 protein had as a result, an efficiency of 108.5%; with slope equal to -3.18 and R2 equal 1.. From a total of 113 samples tested by conventional PCR 63 (55.7%) were positive for rotavirus. From these samples 5 not amplifyed for β-actin gene and were not included in subsequent analyzes. For detection limit of Real-Time PCR was considered the amount of 6x101 copies / reaction, the cut-off being defined cycle (Ct) number 36 to the test with a viral sample from the DNA ligated into plasmid vector. Considering the cut-off 60 (6x101) copies/reaction of the 108 samples tested, 63 (58.3%) were positive to the test. The correlation value obtained by the Kappa test, at a 95% confidence interval, based on results generated between the conventional and PCR testing PCR was 0.279 (low agreement). The results of this study demonstrated that the probe can be efficiently used for a fast and efficient diagnosis of rotavirus of group A, thereby enhancing the repertoire of the established tests
Mozol, Ivan Miletovic. "Análise temporal da expressão gênica e atividade enzimática, relacionadas ao estresse oxidativo e proteômica de Eucalyptus grandis inoculados com Puccinia psidii." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/11/11137/tde-13112013-151232/.
Full textThe eucalyptus planted forest represents the major percentage among all planted forests in Brazil, mainly for the production of paper and cellulose. However, during its development, the eucalyptus can be attacked by a large number of different pathogens, like the fungus Puccinia psidii, the causer of the eucalyptus rust, main disease that affects the eucalyptus in tropical regions. Thus, in order to study some plant defense mechanisms against the infection of the fungus, this study aimed to analyse the gene expression of some enzymes related to oxidative stress and their activities, and the proteome of resistant and susceptible eucalyptus clones, during the process of infection, colonization and multiplication of the fungus in the plant. We could observe differences in the gene expression at all times studied, mostly at 24 hours after inoculation with the fungus. Besides, with the proteome analysis we could find the very relevant proteins for this study, for instance some proteins related to the oxidative stress and to the plant defense response.
Prates, Mirela Cristina Moreira. "Codetecção de bactérias em pacientes com adenoamigdalite crônica." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/17/17151/tde-22062015-152409/.
Full textThe tonsils and adenoids are lymphoid epithelial organs of the upper respiratory tract, where the first contact between inhaled antigens and host defense cells occurs. In fact, the adenoid and tonsillar tissue is in constant contact with a wide variety of bacteria and viruses, which is reflected in the high rate of detection of bacterial and viral pathogens in these tissues, even if healthy. Chronic or recurrent infections of mucosal lymphoid tissues may trigger the development of a chronic inflammatory condition and the presence of tissue hyperplasia. This condition is associated with numerous complications such as rinussinusites repeat, snoring, nasal congestion, Eustachian tube dysfunction, obstructive sleep apnea, otitis media, abnormal facial development, behavioral development. Therefore, this study aims to analyze co-detections of the major human respiratory microbiota bacteria (Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae, Moraxella catahrralis and Pseudomonas aeruginosa) in patients with and without chronic adenoamigdalite using the technique of real time PCR. We had the main bacteria detected in our study H. influenzae, which was found in high levels in the amygdala and adenoids. Already in the secretions, the bacterium most frequently found was S. pneumoniae. In conclusion we can see that the bacteria H. influenzae, M. catarrhalis, and S. pneumoniae had high frequency of detection in amígadalas, adenoids and nasopharyngeal washed children without tonsillar hypertrophy and symptoms of respiratory infection. In addition, there were no major frequency detection of bacteria present in the microbiota in patients with tonsillar hypertrophy than in controls, and unlike the other bacteria, there was moderate to good agreement in the detection of M. catarrhalis and H. influenzae between adenoid and amígdada of individuals without respiratory symptoms.
Stenberg, Jenny. "Optimization and validation of the method lactose intolerance genotyping with real-time PCR." Thesis, Uppsala universitet, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-150810.
Full textBatista, Ribrio Ivan Tavares Pereira. "Efeito do ácido linoléico conjugado TRANS-10, CIS-12 na regulação do acúmulo de lípides e expressão gênica em embriões produzidos in vitro." Universidade Federal de Juiz de Fora (UFJF), 2010. https://repositorio.ufjf.br/jspui/handle/ufjf/2507.
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FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais
A suplementação do ácido linoléico conjugado trans-10, cis-10 no meio de cultivo, representa uma importante alternativa para aumento da sobrevivência dos embriões após a criopreservação. No entanto este isômero de CLA no cultivo in vitro sem antioxidante pode aumentar a taxa apoptótica. O objetivo do presente estudo foi avaliar o efeito da adição CLA trans-10, cis-12 no cultivo in vitro de embriões sem antioxidante. Zigotos bovinos (n = 1.694) foram divididos em dois tratamentos: (T1) grupo controle, zigotos cultivados no meio CR2aa suplementado com soro fetal (n = 815); (T2) zigotos cultivados no meio CR2aa suplementado com soro fetal mais 100 µM CLA trans-10, cis-12. Os embriões foram avaliados quanto a desenvolvimento, quantidade de lípides e criotolerância. Transcritos dos RNA mensageiros (RNAm) dos genes selecionados foram mensurados pelo Real Time PCR. Suplementação de CLA trans-10, cis-12 não afeta significativamente a taxa de blastocisto (31,8% e 34,1% T1 e T2, respectivamente, p = 0,20) e nível dos RNAm dos genes relacionados com stress celular, apoptose e síntese de novo de ácido graxo. Quantidade de lípides e transcritos do RNAm do gene 1-acilglicerol-3-fosfato oaciltransferase 1 enzima relacionado a síntese de triglicérides foram significativamente reduzidos nos embriões cultivados na presença de CLA trans-10, cis-12 em comparação com o grupo controle. Teve aumento significativo na taxa de re-expansão dos blastocistos cultivados na presença de CLA trans-10, cis-12, após o descongelamento (34.4 e 56.3% para T1 e T2, respectivamente p = 0,002). Essa diferença não persistiu na taxa de eclosão (14,0% e 16,5% para T1 e T2, respectivamente, P = 0,62). Esses resultados mostram que o CLA trans-10, cis-12 reduz o acúmulo de lípides nos embriões pela redução nos níveis dos transcritos do gene 1-acilglicerol-3-fosfato o-aciltransferase 1 sem afetar a qualidade do embrião. Adicionalmente, este ácido graxo aumenta a taxa de re-expansão, no entanto, não melhora a taxa de eclosão.
Supplementation of conjugated linoleic acid trans-10, cis-10 in the culture medium, represents an important alternative to increasing the survival of embryos after cryopreservation. However the addition of culture media with CLA trans-10, cis-12 without antioxidant may increase the apoptotic rate. The aim of this study was to evaluate the effect of adding CLA trans-10, cis-12 in vitro culture of embryos without antioxidant. Bovine zygotes (n = 1,694) were divided into two treatments: (T1) control group, zygotes cultured in CR2aa medium supplemented with fetal calf serum (n = 815), (T2) zygotes cultured in CR2aa medium supplemented with fetal calf serum plus 100 µM CLA trans-10, cis-12. Embryos were evaluated for development, amount of lipids and cryotolerance. Transcripts of messenger RNA (mRNA) of selected genes were measured by real time PCR. Supplementation of CLA trans-10, cis-12 did not significantly affect the blastocyst rate (31.8% and 34.1% T1 and T2, respectively, p = 0,20) and the mRNA level of genes related to cell stress, apoptosis and de novo synthesis of fatty acid . Lipids and transcripts of the mRNA of the gene 1-acilglicerol-3-phosphate o-acyltransferase 1 enzyme related to the synthesis of triglycerides were significantly reduced in embryos cultured in the presence of CLA trans-10, cis-12 in comparison the control group. Had increased rate re-expansion of blastocysts cultured in the presence of CLA trans-10, cis-12, after thawing (34.4 and 56.3% for T1 and T2, respectively p = 0,002). This difference did not persist in the hatching rate (14.0 and 16.5% for T1 and T2, respectively, P = 0,62). These results show that the CLA trans-10, cis-12 reduces the accumulation of lipids in the embryos by reducing the levels of gene transcripts acilglicerol-1-3-phosphate oacyltransferase 1 without affecting the quality of the embryo. Additionally, this fatty acid increases the rate re-expansion, but does not improve the hatching rate.
Binder, Michael. "Development of a Botrytis specific immunosensor : towards using PCR species identification." Thesis, Cranfield University, 2014. http://dspace.lib.cranfield.ac.uk/handle/1826/12110.
Full textJungebloud, Anke. "Untersuchung der Genexpression in Aspergillus niger mittels Echtzeit-PCR." Paderborn FIT-Verl. für Innovation und Technologietransfer, 2007. http://www.gbv.de/dms/bs/toc/533996201.pdf.
Full textMangold-Gehring, Sandra. "Bestimmung der Interferon-gamma-Expression bei Baypamune-behandelten Hunden mittels "Real-Time PCR"." [S.l. : s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=97606524X.
Full textThilo, Florian Nikolaus. "Nachweis und Quantifizierung von kolorektalen Tumorzellen in peripherem Blut mittels Real-time PCR." [S.l.] : [s.n.], 2001. http://www.diss.fu-berlin.de/2001/143/index.html.
Full textLorenz, Andreas. "Quantitative Real-time PCR zum spezifischen Nachweis transrenaler DNA des Mycobacterium tuberculosis complex." Diss., lmu, 2010. http://nbn-resolving.de/urn:nbn:de:bvb:19-115143.
Full textSharif, Sanaz. "Comparison of real-time PCR assays for screening of meticillin-resistant Staphylococcus aureus." Thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-154460.
Full textPettersson, Erik. "Investigation of tissue factor mRNA levels in human platelets using real-time PCR." Thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-180831.
Full textUtokaparch, Soraya. "Development of a standardized quantitative real time PCR panel for respiratory viral diagnosis." Thesis, University of British Columbia, 2006. http://hdl.handle.net/2429/31339.
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Medicine, Department of
Experimental Medicine, Division of
Graduate
Gavrilenko, Andreas. "Entwicklung einer real-time multiplex multitube RT-PCR zur Differentialdiagnostik der klassischen Schweinepest." Giessen DVG-Service, 2006. http://deposit.d-nb.de/cgi-bin/dokserv?idn=981170897.
Full textKraemer, Iris. "Untersuchungen zum Vorkommen von Enterobacter sakazakii in Speiseeis mit real-time-PCR-Verfahren." Diss., kostenfrei, 2008. http://edoc.ub.uni-muenchen.de/8377/.
Full textLagmo, Johan. "Development of a multiplex real time PCR assay to target bacteria causing meningitis." Thesis, Uppsala universitet, Klinisk mikrobiologi och infektionsmedicin, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-215641.
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