Navegando por Palavras-chave "Real-Time Polymerase Chain Reaction"
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- ItemSomente MetadadadosDesenvolvimento de PCR em tempo real para detecção rápida de Escherichia coli diarreiogênica(Universidade Federal de São Paulo (UNIFESP), 2012) Souza, Tamara Barros de [UNIFESP]; Scaletsky, Isabel Cristina Affonso [UNIFESP]Introdução: no Brasil, as Escherichia coli ainda estao entre as causas mais importantes de diarreia aguda particularmente em criancas. Com os conhecimentos biologicos, clinicos e epidemiologicos mais aprofundados, foram estabelecidos seis patotipos diferentes de E. coli diarreiogenica (DEC): E. coli enteropatogenica (EPEC), E. coli enterotoxigenica (ETEC), E. coli enteroinvasora (EIEC), E. coli produtora de toxinas de Shiga (STEC) ou E. coli enterohemorragica (EHEC), E. coli enteroagregativa (EAEC) e E. coli que adere difusamente (DAEC). A identificacao dos diferentes patotipos representa um desafio, pois o uso de apenas testes bioquimicos e provas sorologicas nao sao suficientes na deteccao de alguns patotipos. Objetivo: avaliar o desempenho da tecnica da PCR em tempo real para o diagnostico de infeccoes por DEC visando a sua utilizacao na rotina laboratorial. Resultados: inicialmente, foi desenvolvido um ensaio de PCR em tempo real para a deteccao simultanea dos seis patotipos de DEC, e subdivisao de EPEC em tipica e atipica, utilizando o sistema de deteccao SYBR®Green. Este ensaio utiliza nove conjuntos de primers especificos para as sequencias geneticas: eae e pEAF de EPEC, aatA de EAEC, afaBC de DAEC, elt e est de ETEC, ipaH de EIEC e stx1 e stx2 de STEC em uma unica reacao. Posteriormente um segundo ensaio foi desenvolvido para deteccao dos patotipos mais frequentes em nosso meio, EPEC, EAEC e DAEC. Neste ensaio foi escolhido o sistema de deteccao TaqMan® e quatro conjuntos de primers/sondas para detectar a presenca das sequencias geneticas eae e pEAF de EPEC, aatA de EAEC e afaBC de DAEC. Em seguida, a especificidade dos ensaios foi avaliada em amostras comprovadas de DEC, amostras de E. coli nao patogenicas e amostras de outras enterobacterias. Todas as amostras de DEC e de Shigella spp. emitiram sinal de fluorescencia forte e positivo. A sensibilidade foi determinada com as amostras prototipo, e o limite de deteccao foi de aproximadamente 102UFC/mL; em amostras de fezes nao diarreicas inoculadas com diferentes concentracoes de amostras prototipo de DEC, os limites de deteccao variaram de 2,5x107 a 4,5x105UFC/ml por grama de fezes nos ensaios de PCR TaqMan® e PCR SYBR®Green. A aplicacao das PCRs em tempo real foi realizada em amostras de E. coli isoladas das fezes de 97 criancas Quilombolas com diarreia e 285 criancas controle do Espirito Santo, e os resultados comparados com os de PCR convencional e hibridizacao de colonias. Houve total concordancia entre os resultados obtidos com ambos os ensaios, entretanto a PCR SYBR®Green detectou 52 amostras a mais que a PCR convencional e a PCR TaqMan® detectou 96 a mais que a hibridizacao de colonias mas nao identificou 7 amostras. O calculo de coeficiente de Kappa mostrou forte correlacao (0,81) entre a PCR SYBR®Green e a PCR convencional e boa correlacao (0,61) entre a PCR TaqMan® e o teste de hibridizacao de colonias. Conclusao: nossos ensaios de PCR em tempo real oferecem possibilidade de uma rapida e sensivel identificacao simultanea dos diferentes patotipos de DEC, podendo ser utilizados em laboratorios clinicos e de pesquisa
- ItemAcesso aberto (Open Access)Otimização de técnica de PCR em tempo real para detecção das regiões pol e env dos subtipos B e F de HIV-1 e triagem de seus recombinantes(Universidade Federal de São Paulo (UNIFESP), 2009-01-28) Teixeira, Daniela [UNIFESP]; Diaz, Ricardo Sobhie [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Background: The discovery of 42 HIV-1 circulating recombinant forms (CRF) together with the innumerous unique HIV recombinants forms, makes clear the role of genetic recombination for the epidemic. In Brazil, clades B, F, and C co-circulate, with 5 recently described CRFs. Real Time PCR is a rapid and reliable tool capable of detecting different HIV-1 subtypes and recombinant profiles. Objective: The aim of this study was to develop real time PCR systems in order to detect the Brazilian CRF_28 and CRF_29, which are B/F recombinants, as well as detect B/F recombinants generated by in vitro competition assays. In future, these systems should be able to discriminate subtypes in clinical surveys. Methodology: MT-4 cells were separately infected with the viral strains BZ167 (subtype B) and BR020 (subtype F), and supernatant was collected in order to optimizing the real time PCR systems (TaqMan®) developed to detect the subtype profile of different genomic regions, including pol gene (protease, reverse transcriptase, integrase) and env gene (gp120 and gp41). The designed primers should be able to equally amplify the subtype B and F, which should be discriminated by subtype-specific probes. For future validation of these PCR systems, 157 clinical samples from the city of Santos were sequenced and phylogeneticaly analyzed in order to perform the clade assignment with Neighbor-Joining algorithm (Phylip software package v3.5). Results: The designed systems were able to differentiate the utilized viral strains. The estimated efficiencies for each system, for each probe, subtypes B and F separately, were respectively: 80,97 and 85,16% for protease region; 89,80 and 75,09% for reverse transcriptase region; 80,90% and 83,83% for integrase region; 93,49 and 98,93% for gp120 region; and 88,45 and 80,19% for gp41 region. For the co-infected cell culture, the detection of each subtype was performed in the first and fifth passages. Generally, the initial concentration of subtype B appeared to have decreased, some of them becoming undetectable, whereas subtype F seemed to increase with the passages, for protease, reverse transcriptase, gp120 and gp41 regions. The integrase region was an exception, since only the subtype B was detected, with increasing Cycle threshold (Ct) values over time. Sequencing results revealed that 65 out of 157 samples had the subtype profile defined for all regions. 43 out of 71 were defined as B, whereas 3 were F in all regions. 12 samples presented the CRF_28 profile, and 9 samples presented the CRF_29 profile. There were no subtype C samples in any genomic regions analyzed. Conclusion: The use of real time PCR technique for identification of fragments’ subtypes in cell culture and for evaluation of replicative dynamics of recombination in co-infected cultures warrants its potential use in future in vivo surveys. This methodology proved to be efficient, fast, less cumbersome and less expensive than DNA sequencing. The newly designed systems performed for supernatant of competition assays had suggested a divergent distribution of subtypes for the different regions, which reflects the possibility of genetic recombination. Results from clinical samples revealed a high prevalence of CRF_28/29 in this geographic region, thus reflecting the resulting consequence of different co-circulating strains and pointing to the need for a careful surveillance.
- ItemAcesso aberto (Open Access)Oxidative stress gene expression profile in inbred mouse after ischemia/reperfusion small bowel injury(Sociedade Brasileira para o Desenvolvimento da Pesquisa em Cirurgia, 2012-11-01) Bertoletto, Paulo Roberto; Ikejiri, Adauto Tsutomu; Somaio Neto, Frederico; Chaves, José Carlos; Teruya, Roberto; Bertoletto, Eduardo Rodrigues; Taha, Murched Omar [UNIFESP]; Fagundes, Djalma José [UNIFESP]; UFGD Medical School; Federal University of Mato Grosso do Sul; Universidade Federal de São Paulo (UNIFESP)PURPOSE: To determine the profile of gene expressions associated with oxidative stress and thereby contribute to establish parameters about the role of enzyme clusters related to the ischemia/reperfusion intestinal injury. METHODS: Twelve male inbred mice (C57BL/6) were randomly assigned: Control Group (CG) submitted to anesthesia, laparotomy and observed by 120min; Ischemia/reperfusion Group (IRG) submitted to anesthesia, laparotomy, 60min of small bowel ischemia and 60min of reperfusion. A pool of six samples was submitted to the qPCR-RT protocol (six clusters) for mouse oxidative stress and antioxidant defense pathways. RESULTS: On the 84 genes investigated, 64 (76.2%) had statistic significant expression and 20 (23.8%) showed no statistical difference to the control group. From these 64 significantly expressed genes, 60 (93.7%) were up-regulated and 04 (6.3%) were down-regulated. From the group with no statistical significantly expression, 12 genes were up-regulated and 8 genes were down-regulated. Surprisingly, 37 (44.04%) showed a higher than threefold up-regulation and then arbitrarily the values was considered as a very significant. Thus, 37 genes (44.04%) were expressed very significantly up-regulated. The remained 47 (55.9%) genes were up-regulated less than three folds (35 genes - 41.6%) or down-regulated less than three folds (12 genes - 14.3%). CONCLUSION: The intestinal ischemia and reperfusion promote a global hyper-expression profile of six different clusters genes related to antioxidant defense and oxidative stress.
- ItemSomente MetadadadosPesquisa De Podocitúria: Desenvolvimento E Padronização De Métodos(Universidade Federal de São Paulo (UNIFESP), 2018-02-22) Pereira, Amelia Rodrigues [UNIFESP]; Kirsztajn, Gianna Mastroianni [UNIFESP]Introduction: Podocytes Are Fundamental Cells For Maintaining The Functionality Of The Glomerular Filtration Barrier, Which Is Characterized By Selective Permeability. Podocyturia, Excretion Of Podocytes In Urine, Has Been Studied In Several Glomerulopathies As A Potential Marker Of Disease Activity. It Is A Non-Invasive Method That Can Reflect In Real Time The Podocyte Lesion, And It Can Be A More Sensitive And Earlier Marker Of This Kind Of Lesion. It Presents Potential Applicability In Assessing The Activity Of Glomerular Diseases And Understanding Their Pathogenesis, As Well As Guiding Future Therapeutic Interventions. Objectives: To Standardize And Validate The Methodology For The Investigation Of Podocyturia By Two Techniques, Indirect Immunofluorescence And Quantification By Polymerase Chain Reaction In Real Time, In Addition To Determine Which Technique Is Most Suitable For This Purpose. Material And Methods: The Study Of Podocyturia Occurred In A Group Of 71 Individuals, 21 (29,17%) Patients With Foc
- ItemAcesso aberto (Open Access)Pesquisa de Polymavirus-BK em transplantes renais: incidência e correlação com parâmetros clínicos e laboratoriais(Universidade Federal de São Paulo (UNIFESP), 2010-08-25) Svicero, Bianca Silva [UNIFESP]; Granato, Celso Francisco Hernandes [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Introduction: The BK virus (BKV) belongs to the Polyomaviridae family, is a non enveloped virus with double-stranded DNA, may be associated with hemorrhagic cystitis, ureteral stenosis, and in some cases, cancer. The polyomaviruses are found in about 80% of the general population, however its clinical manifestations are rare and limited to individuals with weakened immune system. The BKV is a virus with the potential to establish latency and reactivate. Asymptomatic reactivation and low level of replication are observed in approximately 5% of the healthy population. In the patients of renal transplant. Reactivation becomes relevant, since 10-40% of patients with BKV active infection can progress to graft loss. The diagnosis of BKV infection can be accomplished by “decoy” cells in urinary sediment and / or viral load evaluation by polymerase chain reaction in Real Time (qPCR). Treatment is not defined by antiviral drugs but by reducing the dose of immunosuppressive drugs. Objectives: The aim of this study was to standardize Real Time PCR to estimate the BKV viral load in plasma and urine and the presence of “decoy” cells in urine of patients undergoing renal transplantation in the period between november/2008 and may/2010, since pre transplant until 12 months after transplantation. Analysis of epidemiological aspects related to infection and the correlation with the course of the disease were evaluated as well. Methods: Analysis of urine sediment by Papanicolaou staining for the presence of "decoy" cells by electron microscopy. For standardization of Real-Time PCR in plasma and urine a commercial BKV DNA with a viral load set at 1.6 x105cópias / mL was used for the construction of standard curve; betaglobin was used as an internal control. Based in the techinique publishe in 2004 by Randhawa and colleagues. Results: The study included 81 patients followed prospectively over 12 months, where 70.3% were male with median age of 39 years at the time of transplantation. The urine cytology provided a positive result in 1.6% of the samples. The standard curve was performed by five serial dilutions of positive control (1-104), the slope of the curve was -3.60, the Pearson correlation coefficient was r2 = 0.99 and efficiency of the reaction was 91 6%. Plasma samples showed variations in viral load, 39.1% from the 1.08 x104 9.51 x104 copies / mL and 26.3% with viral load greater than or equal to 1x105 copies / mL and 34.6% with viral load below 1x104 copies / mL. We demonstrated an incidence of 29 cases by 1000 patients. The clinical features did not correlate with laboratory findings found, since there was no description of symptoms related to infection with BKV in patients supposedly with viral load indicative of infection during the 12 months analyzed. Conclusions: The standardization of real-time PCR was achieved and can be compared with previous studies..We didn’t consider the PCR results indicative of disease because a reanl biopsy was not performed. The result of decoy cells research was confirmed by another analyst who was unaware of the survey data. We couldn’t find define another laboratory parameters as predictors of infection by BKV.