[Application of denaturing high perfomance liquid chromatography in detection of rpoB mutations associated with rifampin resistance in Mycobacterium tuberculosis].

Miao Li, Hong Yan Si, L. Guo, Pei Qiang Li, Bing Dong Zhu, Ke Hu Yang, Ying Zhang, Tao Jing

Research output: Contribution to journalArticle

Abstract

to explore the value of denaturing high performance liquid chromatography (DHPLC) in detection of rpoB mutations in rifampin-resistant M. tuberculosis, in order to establish a convenient and rapid approach to screening rpoB gene mutations in M. tuberculosis. rifampin resistance-determining region of rpoB gene in M. tuberculosis was amplified by PCR and further analyzed by DHPLC to screen mutations at optimized denaturation temperature (65.4 degrees C), which utilized heteroduplex formation between wild-type and mutated DNA strands to identify mutations. The PCR products from strains with different chromatographic profiles were sequenced further to evaluate the sensitivity and specificity. there were 46 M. tuberculosis strains including 42 rifampin resistance strains and 4 rifampin sensitive strains. From these strains, 15 different chromatographic profiles were produced by DHPLC. Combined with the results of gene sequencing, it was shown that strains with different chromatographic profiles had distinct mutations. Except D108 and D24 which had same chromatographic profiles but different genetic polymorphisms, all other strains showed consistent chromatographic profiles with genetic polymorphisms, i.e. if they had identical chromatographic profiles, their genetic polymorphism were the same; but if they had different chromatographic profiles, their genetic polymorphism were also different. DHPLC is a simple, efficient, high-throughput and automatic method with high sensitivity and specificity, which may be useful in the rapid detection of rpoB gene mutation in M. tuberculosis.

Original languageEnglish (US)
Pages (from-to)128-131
Number of pages4
JournalZhonghua jie he he hu xi za zhi = Zhonghua jiehe he huxi zazhi = Chinese journal of tuberculosis and respiratory diseases
Volume33
Issue number2
StatePublished - Feb 2010
Externally publishedYes

Fingerprint

Rifampin
Mycobacterium tuberculosis
Liquid Chromatography
Genetic Polymorphisms
Mutation
High Pressure Liquid Chromatography
Genes
Sensitivity and Specificity
Polymerase Chain Reaction
Temperature
DNA

ASJC Scopus subject areas

  • Pulmonary and Respiratory Medicine

Cite this

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title = "[Application of denaturing high perfomance liquid chromatography in detection of rpoB mutations associated with rifampin resistance in Mycobacterium tuberculosis].",
abstract = "to explore the value of denaturing high performance liquid chromatography (DHPLC) in detection of rpoB mutations in rifampin-resistant M. tuberculosis, in order to establish a convenient and rapid approach to screening rpoB gene mutations in M. tuberculosis. rifampin resistance-determining region of rpoB gene in M. tuberculosis was amplified by PCR and further analyzed by DHPLC to screen mutations at optimized denaturation temperature (65.4 degrees C), which utilized heteroduplex formation between wild-type and mutated DNA strands to identify mutations. The PCR products from strains with different chromatographic profiles were sequenced further to evaluate the sensitivity and specificity. there were 46 M. tuberculosis strains including 42 rifampin resistance strains and 4 rifampin sensitive strains. From these strains, 15 different chromatographic profiles were produced by DHPLC. Combined with the results of gene sequencing, it was shown that strains with different chromatographic profiles had distinct mutations. Except D108 and D24 which had same chromatographic profiles but different genetic polymorphisms, all other strains showed consistent chromatographic profiles with genetic polymorphisms, i.e. if they had identical chromatographic profiles, their genetic polymorphism were the same; but if they had different chromatographic profiles, their genetic polymorphism were also different. DHPLC is a simple, efficient, high-throughput and automatic method with high sensitivity and specificity, which may be useful in the rapid detection of rpoB gene mutation in M. tuberculosis.",
author = "Miao Li and Si, {Hong Yan} and L. Guo and Li, {Pei Qiang} and Zhu, {Bing Dong} and Yang, {Ke Hu} and Ying Zhang and Tao Jing",
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T1 - [Application of denaturing high perfomance liquid chromatography in detection of rpoB mutations associated with rifampin resistance in Mycobacterium tuberculosis].

AU - Li, Miao

AU - Si, Hong Yan

AU - Guo, L.

AU - Li, Pei Qiang

AU - Zhu, Bing Dong

AU - Yang, Ke Hu

AU - Zhang, Ying

AU - Jing, Tao

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