Análise de diferentes primers utilizados na PCR visando ao diagnóstico da tuberculose no Estado do Amazonas

Abstract

Background: Various primers are being tested for the detection of Mycobacterium tuberculosis DNA. The accuracy of the polymerase chain reaction (PCR) depends on the target sequence used and whether the test will be performed in culture or in clinical specimens. Objectives:To identify DNA sequences, specifically those commonly reported as targets for diagnosis of tuberculosis (TB), in clinical samples of M. tuberculosis strains.Method: Eighty-one clinical samples from suspected TB patients were initially processed and submitted to bacilloscopy (smear) and culture, and PCR was performed with specific primers for the following targets: IS6110, 65 kDa, 38 kDa and MPB64.Results: Smear and culture results were negative in 24 samples, as was the PCR. The 19 samples testing smear positive, as well as the isolated strains, were 100% positive on PCR, with the exception of the 89.4% result from PCR with MPB64 primers. In the 38 smear negative and culture positive samples, PCR results were inconsistent. The primers specific for amplifying the 123 bp IS6110 fragment gave the highest positivity (92.1%), diagnostic agreement (0.9143), co-positivity (94.7%) and co-negativity (100%). Conclusion:The IS6110, 38 kDa, MPB64 and 65 kDa sequences were found in the genome of all M. tuberculosis strains isolated in patients from the state of Amazonas. The protocol for processing the clinical samples prior to PCR analysis and the specific primers used to amplify the 123bp IS6110 fragment showed a greater efficiency in diagnosing pulmonary (paucibacillary) tuberculosis in comparison to published data.

HIV-associated tuberculous lymphadenitis: the importance of polymerase chain reaction (PCR) as a complementary tool for the diagnosis of tuberculosis - a study of 104 patients

“…16 When nested PCR was used to identify MTB positivity in skin biopsies of suspected cutaneous TB, PCR positivity in ZiehlNeelsen positive samples was 66%. 15 The present results suggest a positivity (sensitivity) of 63% when compared with a combination of Lowenstein-Jensen culture and histopathology ( Since a conventional, low-cost PCR was used in this study, it is likely that inhibition or even a low detection range of the PCR reaction might be the cause. Optimizing a real-time PCR reaction using primers and probes in this region may improve mean sensitivity rates and also increase detection of resistant forms in multiplex assays.…”

“…[9][10][11][12] In this regard, in-house conventional polymerase chain reaction (PCR) based on amplifying the IS6110 insertion element can be used to amplify Mycobacterium tuberculosis (MTB) DNA, offering the potential of a sensitive, specific and rapid diagnostic tool for either ruling out or considering pulmonary or extra pulmonary tuberculosis. [13][14][15] The present study was conducted in a hospital setting in the Amazonas Foundation of Tropical Medicine [Fundação de Medicina Tropical Amazonas (FMTAM)], where the incidence of both TB and HIV is high. A protocol was designed to investigate the effectiveness of combining PCR with histopathological examination, Ziehl-Neelsen staining and culture in Lowenstein-Jensen medium to diagnose TB using lymph node excisional biopsies.…”

Nested-PCR do gene que codifica o antígeno b aplicada ao diagnóstico da tuberculose pulmonar

“…Os autores relatam que em amostras clínicas multibacilares, apenas a seqüência MPB64 não apresentou 100% de eficácia para diagnóstico da tuberculose, sendo observado 89% de detecção dos casos positivos. Entre as amostras paucibacilares a co-positividade foi variada, no entanto, o fragmento de 123pb do alvo IS6110 apresentou 95% de co-positividade com a cultura, resultado superior aos demais marcadores, incluindo o gene da proteína de 38kDa (91%) avaliado naquele estudo 11 . Para Ogusku & Salem (2004) a maior eficácia da seqüência IS6110 para o diagnóstico da tuberculose em relação ao gene codificante da proteína 38kDa pode estar associada ao fato de o primeiro estar presente em múltiplas cópias no genoma do M. tuberculosis (5 a 19 cópias).…”

Avaliação da reação em cadeia da polimerase no diagnóstico da tuberculose pulmonar em pacientes indígenas e não indígenas

“…The DNA of the sputum samples, decontaminated and stored at -20ºC, as well as the DNA of the M. tuberculosis strains isolated, was extracted according to the recommendations of Ogusku and Salem. (9) In each session, negative controls for DNA extraction were included, in order to rule out the possibility of cross-contamination among the samples.…”