Locked Nucleic Acid Probe-Based Real-Time PCR Assay for the Rapid Detection of Rifampin-Resistant <i>Mycobacterium tuberculosis</i>

<div><p>Drug-resistant <i>Mycobacterium tuberculosis</i> 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 <i>rpoB</i> gene associated with rifampin (RFP) resistance in <i>M</i>. <i>tuberculosis</i>. Six LNA probes with the discrimination capability of one-base mismatch were designed to monitor the 23 most frequent <i>rpoB</i> mutations. The target mutations were identified using the probes in a “probe dropout” manner (quantification 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 to <i>M</i>. <i>tuberculosis</i> with or without RFP resistance by evaluating 12 strains of common non-tuberculosis mycobacteria. The limit of detection of <i>M</i>. <i>tuberculosis</i> was 10 genomic equivalents (GE)/reaction by further introducing a nested PCR method. In a blind validation of 154 clinical mycobacterium isolates, 142/142 (100%) were correctly detected through the assay. Of these isolates, 88/88 (100%) were determined as RFP susceptible and 52/54 (96.3%) were characterized as RFP resistant. Two unrecognized RFP-resistant strains were sequenced and were found to contain mutations outside the range of the 23 mutation targets. In conclusion, this study established a sensitive, accurate, and low-cost LNA probe-based assay suitable for a four-multiplexing real-time PCR instrument. The proposed method can be used to diagnose RFP-resistant tuberculosis in clinical laboratories.</p>