Direct genotypic detection of Mycobacterium tuberculosis rifampin resistance in clinical specimens by using single-tube heminested PCR

Author:

Whelen A C1,Felmlee T A1,Hunt J M1,Williams D L1,Roberts G D1,Stockman L1,Persing D H1

Affiliation:

1. Division of Clinical Microbiology, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.

Abstract

Recent analysis of the gene encoding the beta subunit of Mycobacterium tuberculosis RNA polymerase (rpoB) has demonstrated a small region that harbors the mutations most frequently associated with rifampin resistance. Earlier reports have described a high degree of sequence conservation of rpoB among mycobacteria other than M. tuberculosis and other GC-rich bacteria that can lead to false-positive amplification when applied directly to clinical specimens. We developed reagents for PCR amplification that are based on signature nucleotides discovered by comparative sequence analysis of the rpoB genes of organisms phylogenetically related to M. tuberculosis. The specificities of the reagents were challenged with 20 isolates of multiple-drug-resistant M. tuberculosis and more than 20 species of mycobacteria other than M. tuberculosis and other GC-rich organisms. A single-tube heminested PCR protocol was devised to obtain sensitivity equal to those of an IS6110-based PCR assay and culture in spiked sputum experiments. The assay correctly identified 21 of 24 (87.5%) culture-positive specimens, 13 of which were acid-fast smear-negative, in a panel of 51 clinical specimens. Three specimens that were false-positive initially were negative upon repeat testing when the assay was modified to eliminate the potential for aerosol carryover of the first-round amplification product during the open-tube addition of the second set of reaction reagents. This assay is the most sensitive and specific test to date for the direct detection of M. tuberculosis rpoB in clinical specimens. This rapid PCR-based assay can be used for the simultaneous identification of M. tuberculosis and its rifampin susceptibility genotype.

Publisher

American Society for Microbiology

Subject

Microbiology (medical)

Reference32 articles.

1. Nationwide survey of drug-resistant tuberculosis in the United States;Bloch A. B.;JAMA,1994

2. Detection and identification of mycobacteria by amplification of rRNA;Boeddinghaus B.;J. Clin. Microbiol.,1990

3. Rapid and simple method for purification of nucleic acids;Boom R.;J. Clin. Microbiol.,1990

4. Bottger E. C. A. Meier P. Kirschner F. Bange and U. Vogel. 1994. Molecular basis of drug resistance in Mycobacteria abstr. U-124 p. 194. In Abstracts of the 94th General Meeting of the American Society for Microbiology 1994. American Society for Microbiology Washington D.C.

5. Post-PCR sterilization: a method to control carryover contamination for the polymerase chain reaction;Cimino G. D.;Nucleic Acids Res.,1991

Cited by 63 articles. 订阅此论文施引文献 订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献

1. PCR and Its Variations;Advanced Techniques in Diagnostic Microbiology;2018

2. Detection of novel and reported mutations in the rpoB, katG and inhA genes in multidrug-resistant tuberculosis isolates: A hospital-based study;Journal of Global Antimicrobial Resistance;2015-03

3. PCR and Its Variations;Advanced Techniques in Diagnostic Microbiology;2012-08-06

4. Rheonix CARD® Technology;Point of Care: The Journal of Near-Patient Testing & Technology;2012-03

5. Detection of isoniazid and rifampin resistance of Mycobacterium tuberculosis by a multiplex allele-specific polymerase chain reaction (PCR) assay;International Journal of Mycobacteriology;2012-03

同舟云学术

1.学者识别学者识别

2.学术分析学术分析

3.人才评估人才评估

"同舟云学术"是以全球学者为主线,采集、加工和组织学术论文而形成的新型学术文献查询和分析系统,可以对全球学者进行文献检索和人才价值评估。用户可以通过关注某些学科领域的顶尖人物而持续追踪该领域的学科进展和研究前沿。经过近期的数据扩容,当前同舟云学术共收录了国内外主流学术期刊6万余种,收集的期刊论文及会议论文总量共计约1.5亿篇,并以每天添加12000余篇中外论文的速度递增。我们也可以为用户提供个性化、定制化的学者数据。欢迎来电咨询!咨询电话:010-8811{复制后删除}0370

www.globalauthorid.com

TOP

Copyright © 2019-2024 北京同舟云网络信息技术有限公司
京公网安备11010802033243号  京ICP备18003416号-3