RESEARCH ARTICLE Locked Nucleic Acid Probe-Based Real-Time PCR Assay for the Rapid Detection of Rifampin-ResistantMycobacterium

Drug-resistantMycobacterium tuberculosis can be rapidly diagnosed through nucleic acid amplification techniques by analyzing the variations in the associated gene sequences. In the present study, a locked nucleic acid (LNA) probe-based real-time PCR assay was developed to identify the mutations in the rpoB gene associated with rifampin (RFP) resistance inM. tuberculosis. Six LNA probes with the discrimination capability of one-base mismatch were designed to monitor the 23 most frequent rpoBmutations. The target mutations were identified using the probes in a “probe dropout ” manner (quantifica-tion cycle = 0); thus, the proposed technique exhibited superiority in mutation detection. The LNA probe-based real-time PCR assay was developed in a two-tube format with three LNA probes and one internal amplification control probe in each tube. The assay showed excellent specificity toM. tuberculosis with or without RFP resistance by evaluat-ing 12 strains of common non-tuberculosis mycobacteria. The limit of detection ofM. tuberculosis was 10 genomic equivalents (GE)/reaction by further introducing a neste