title: 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: National Center for Tuberculosis Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China.
authors: Yu
authors: Y
authors: Pang
authors: Guan
authors: G
authors: Liu
authors: Yufeng
authors: Y
authors: Wang
authors: Suhua
authors: S
authors: Zheng
authors: Yan-Lin
authors: YL
authors: Zhao
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology; D004269:DNA, Bacterial / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D024881:Drug Resistance, Bacterial; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012293:Rifampin / Q000494:pharmacology; D012680:Sensitivity and Specificity; D044366:Transition Temperature
chemical_list: D000995:Antitubercular Agents; D004269:DNA, Bacterial; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit; D012293:Rifampin