Integrated Microfluidic Card with TaqMan Probes and High-Resolution Melt Analysis To Detect Tuberculosis Drug Resistance Mutations across 10 Genes

ABSTRACT Genotypic methods for drug susceptibility testing of Mycobacterium tuberculosis are desirable to speed the diagnosis and proper therapy of tuberculosis (TB). However, the numbers of genes and polymorphisms implicated in resistance have proliferated, challenging diagnostic design. We develop...

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Autores principales:	Suporn Pholwat, Jie Liu, Suzanne Stroup, Jean Gratz, Sayera Banu, S. M. Mazidur Rahman, Sara Sabrina Ferdous, Suporn Foongladda, Duangjai Boonlert, Oleg Ogarkov, Svetlana Zhdanova, Gibson Kibiki, Scott Heysell, Eric Houpt
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2015
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/40c89c90aba94a38ab5b5575d00bf941
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spelling	oai:doaj.org-article:40c89c90aba94a38ab5b5575d00bf9412021-11-15T15:41:33ZIntegrated Microfluidic Card with TaqMan Probes and High-Resolution Melt Analysis To Detect Tuberculosis Drug Resistance Mutations across 10 Genes10.1128/mBio.02273-142150-7511https://doaj.org/article/40c89c90aba94a38ab5b5575d00bf9412015-05-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02273-14https://doaj.org/toc/2150-7511ABSTRACT Genotypic methods for drug susceptibility testing of Mycobacterium tuberculosis are desirable to speed the diagnosis and proper therapy of tuberculosis (TB). However, the numbers of genes and polymorphisms implicated in resistance have proliferated, challenging diagnostic design. We developed a microfluidic TaqMan array card (TAC) that utilizes both sequence-specific probes and high-resolution melt analysis (HRM), providing two layers of detection of mutations. Twenty-seven primer pairs and 40 probes were designed to interrogate 3,200 base pairs of critical regions of the inhA, katG, rpoB, embB, rpsL, rrs, eis, gyrA, gyrB, and pncA genes. The method was evaluated on 230 clinical M. tuberculosis isolates from around the world, and it yielded 96.1% accuracy (2,431/2,530) in comparison to that of Sanger sequencing and 87% accuracy in comparison to that of the slow culture-based susceptibility testing. This TAC-HRM method integrates assays for 10 genes to yield fast, comprehensive, and accurate drug susceptibility results for the 9 major antibiotics used to treat TB and could be deployed to improve treatment outcomes. IMPORTANCE Multidrug-resistant tuberculosis threatens global tuberculosis control efforts. Optimal therapy utilizes susceptibility test results to guide individualized treatment regimens; however, the susceptibility testing methods in use are technically difficult and slow. We developed an integrated TaqMan array card method with high-resolution melt analysis that interrogates 10 genes to yield a fast, comprehensive, and accurate drug susceptibility result for the 9 major antituberculosis antibiotics.Suporn PholwatJie LiuSuzanne StroupJean GratzSayera BanuS. M. Mazidur RahmanSara Sabrina FerdousSuporn FoongladdaDuangjai BoonlertOleg OgarkovSvetlana ZhdanovaGibson KibikiScott HeysellEric HouptAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 6, Iss 2 (2015)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Suporn Pholwat Jie Liu Suzanne Stroup Jean Gratz Sayera Banu S. M. Mazidur Rahman Sara Sabrina Ferdous Suporn Foongladda Duangjai Boonlert Oleg Ogarkov Svetlana Zhdanova Gibson Kibiki Scott Heysell Eric Houpt Integrated Microfluidic Card with TaqMan Probes and High-Resolution Melt Analysis To Detect Tuberculosis Drug Resistance Mutations across 10 Genes
description	ABSTRACT Genotypic methods for drug susceptibility testing of Mycobacterium tuberculosis are desirable to speed the diagnosis and proper therapy of tuberculosis (TB). However, the numbers of genes and polymorphisms implicated in resistance have proliferated, challenging diagnostic design. We developed a microfluidic TaqMan array card (TAC) that utilizes both sequence-specific probes and high-resolution melt analysis (HRM), providing two layers of detection of mutations. Twenty-seven primer pairs and 40 probes were designed to interrogate 3,200 base pairs of critical regions of the inhA, katG, rpoB, embB, rpsL, rrs, eis, gyrA, gyrB, and pncA genes. The method was evaluated on 230 clinical M. tuberculosis isolates from around the world, and it yielded 96.1% accuracy (2,431/2,530) in comparison to that of Sanger sequencing and 87% accuracy in comparison to that of the slow culture-based susceptibility testing. This TAC-HRM method integrates assays for 10 genes to yield fast, comprehensive, and accurate drug susceptibility results for the 9 major antibiotics used to treat TB and could be deployed to improve treatment outcomes. IMPORTANCE Multidrug-resistant tuberculosis threatens global tuberculosis control efforts. Optimal therapy utilizes susceptibility test results to guide individualized treatment regimens; however, the susceptibility testing methods in use are technically difficult and slow. We developed an integrated TaqMan array card method with high-resolution melt analysis that interrogates 10 genes to yield a fast, comprehensive, and accurate drug susceptibility result for the 9 major antituberculosis antibiotics.
format	article
author	Suporn Pholwat Jie Liu Suzanne Stroup Jean Gratz Sayera Banu S. M. Mazidur Rahman Sara Sabrina Ferdous Suporn Foongladda Duangjai Boonlert Oleg Ogarkov Svetlana Zhdanova Gibson Kibiki Scott Heysell Eric Houpt
author_facet	Suporn Pholwat Jie Liu Suzanne Stroup Jean Gratz Sayera Banu S. M. Mazidur Rahman Sara Sabrina Ferdous Suporn Foongladda Duangjai Boonlert Oleg Ogarkov Svetlana Zhdanova Gibson Kibiki Scott Heysell Eric Houpt
author_sort	Suporn Pholwat
title	Integrated Microfluidic Card with TaqMan Probes and High-Resolution Melt Analysis To Detect Tuberculosis Drug Resistance Mutations across 10 Genes
title_short	Integrated Microfluidic Card with TaqMan Probes and High-Resolution Melt Analysis To Detect Tuberculosis Drug Resistance Mutations across 10 Genes
title_full	Integrated Microfluidic Card with TaqMan Probes and High-Resolution Melt Analysis To Detect Tuberculosis Drug Resistance Mutations across 10 Genes
title_fullStr	Integrated Microfluidic Card with TaqMan Probes and High-Resolution Melt Analysis To Detect Tuberculosis Drug Resistance Mutations across 10 Genes
title_full_unstemmed	Integrated Microfluidic Card with TaqMan Probes and High-Resolution Melt Analysis To Detect Tuberculosis Drug Resistance Mutations across 10 Genes
title_sort	integrated microfluidic card with taqman probes and high-resolution melt analysis to detect tuberculosis drug resistance mutations across 10 genes
publisher	American Society for Microbiology
publishDate	2015
url	https://doaj.org/article/40c89c90aba94a38ab5b5575d00bf941
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Integrated Microfluidic Card with TaqMan Probes and High-Resolution Melt Analysis To Detect Tuberculosis Drug Resistance Mutations across 10 Genes

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