Real time PCR quantification of viable Mycobacterium tuberculosis from sputum samples treated with propidium monoazide

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• 作者：Thiago Milech de Assunç ; ã ; o^a ; ^b ; ¹ ; Eraldo L. Batista Jr.^c ; Candida Deves^a ; ^d ; ¹ ; Anne Drumond Villela^a ; ^d ; ¹ ; Vany Elisa Pagnussatti^e ; ^f ; Ana Christina de Oliveira Dias^a ; ¹ ; Afrâ ; nio Kritski^g ; Valnê ; s Rodrigues-Junior^a ; ^d ; ^{valnes.rodrigues@acad.pucrs.br" class="auth_mail} ; Luiz Augusto Basso^a ; ^b ; ^d ; ¹ ; Dió ; genes Santiago Santos^a ; ^b ; ^{diogenes@pucrs.br" class="auth_mail}
• 关键词：Diagnosis ; Tuberculosis ; Real time PCR ; Smear ; PMA
• 刊名：Tuberculosis
• 出版年：July 2014
• 年：2014
• 卷：94
• 期：4
• 页码：421-427
• 全文大小：682 K

文摘

Diagnostic methods of TB, nowadays, are prone to delay in diagnosis, increased false negative results and are not sensitive to many forms of paucibacillary disease. The aims of this study were to implement a quantitative nucleic acid-based diagnostic test for paucibacillary tuberculosis, enabling the identification and quantification of viable m>Mycobacterium tuberculosism> bacilli by quantitative Real-Time PCR (qRT-PCR). The intergenic region of the single-copy m>inhA-mabAm> gene was chosen as the target region for design of primers and probes conjugated with fluorophores. The construction of synthetic DNA flanking the target region served as standards for absolute quantification of nucleic acids. Using the intercaling dye, propidium monoazide, we were able to discriminate between viable and dead cells of m>M. tuberculosism>. The diagnosis method showed a broad sensitivity (96.1%) when only compared to samples of smear-positive sputum and ROC analyses shows that our approach performed well and yielded a specificity of 84.6% and a sensitivity of 84.6% when compared to m>M. tuberculosism> colony-forming units counting.