Combining COLD-PCR and high-resolution melt analysis for rapid detection of low-level, rifampin-resistant mutations in Mycobacterium tuberculosis.

Abstract

Multidrug-resistant Mycobacterium tuberculosis (M. tuberculosis) remains a serious threat to public health. Mutational analysis of the gene encoding the beta subunit of RNA polymerase (rpoB) is an established and widely used surrogate marker for multidrug-resistant tuberculosis (MDR-TB). The rpoB-based drug-resistant assay requires relatively less time to detect drug resistance in M. tuberculosis, yet it fails to detect low-level mutations in wild-type DNA. Here, we describe a low-level mutation detection method that combines co-amplification at lower denaturation temperature polymerase chain reaction (COLD-PCR) with high-resolution melting (HRM) analysis, aimed at detecting low-level, rifampin-resistant mutations in M. tuberculosis. Compared to conventional polymerase chain reaction (PCR), dilution experiments demonstrated a four- to eightfold improvement in selectivity using COLD-PCR/HRM to detect low-level, rifampin-resistant mutations. The mutation detection limit of conventional PCR/HRM was approximately 20%, whereas COLD-PCR/HRM had a mutation detection limit of 2.5%. Using traditional PCR/HRM and DNA sequencing, we found rpoB mutation in 110 rifampin-resistant isolates. The use of COLD-PCR/HRM allowed us to detect 10 low-level, rifampin-resistant mutations in 16 additional drug-resistant isolates. The sensitivity of COLD-PCR/HRM (95.2%) is significantly higher than that of PCR/HRM (87.3%). Our findings demonstrate that combined use of COLD-PCR with HRM can provide a sensitivity of at least 5% in detecting rpoB-mutated populations in a wild-type background, decreasing the delay in drug-resistant TB diagnosis and leading to faster, cheaper, more efficient, and more personalized antibiotic treatment, especially for low-level drug resistance mutations among the excess wild-type DNA.

• Authors: Yu Pang, Guan Liu, Yufeng Wang, Suhua Zheng, Yan-Lin Zhao
• Journal: Journal of microbiological methods (J Microbiol Methods) Vol. 93 Issue 1 Pg. 32-6 (Apr 2013) ISSN: 1872-8359 [Electronic] Netherlands
• PMID: 23396215 (Publication Type: Evaluation Study, Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2013 Elsevier B.V. All rights reserved.

Chemical References

• Antitubercular Agents
• DNA, Bacterial
• DNA-Directed RNA Polymerases
• RNA polymerase beta subunit
• Rifampin

Topics

• Antitubercular Agents (pharmacology)
• DNA, Bacterial (genetics)
• DNA-Directed RNA Polymerases (genetics)
• Drug Resistance, Bacterial
• Mutation
• Mycobacterium tuberculosis (drug effects, genetics)
• Nucleic Acid Denaturation
• Polymerase Chain Reaction (methods)
• Rifampin (pharmacology)
• Sensitivity and Specificity
• Transition Temperature