Abstract:

Objective To establish a probe-directed recombinase amplification (PDRA) assay for rapid detection of 516 mutations in Mycobacterium tuberculosis (MTB) rpoB gene. Methods Four probes including one wild-type (P-W) probe and three mutant-type probes (P-GGC, P-GTC, P-TAC) and a common downstream primer were designed to construct four detection systems (TB-516-W, TB-516-GGC, TB-516-GTC, and TB-516-TAC), respectively. The isothermal amplification was performed at 39 ℃ for 40 min. The positive threshold time was used to determine the occurrence and type of mutation at 516 locus of the MTB rpoB gene. The sensitivity of the detection system was assessed by detecting the corresponding recombinant plasmid with the same probe. The specificity of the detection system was assessed by detecting the same recombinant plasmid with different probes. The established PDRA assay was used to detect 6 MTB rifampicin-resistant strains and 35 MTB culture-positive sputum specimens, and compared with the results of Sanger sequencing. Results The sensitivity of the four detection systems for corresponding recombinant plasmids was 1000 copies/μl. When the probe and the target sequence were completely matched, the positive threshold time was short; otherwise, the positive threshold time was long, with a stable difference in positive threshold time, suggesting the good specificity of the four detection systems. The positive threshold time differences of the systems TB-516-W, TB-516-GGC, TB-516-GTC and TB-516-TAC were 7.0, 5.8, 10.0 and 7.0 min, respectively. The PDRA detection results of 6 MTB-resistant strains and 35 MTB culture-positive sputum samples were consistent with the sequencing results. Conclusion This study has preliminarily established a PDRA assay that can be applied to detect the 516 mutations in rpoB gene of MTB. It has high detection sensitivity and good specificity, and therefore has certain laboratory application value.

Key words: Nucleic acid amplification techniques, Recombinases, Mycobacterium tuberculosis, Genes