Innovation
Focusing on the research and development and application of isothermal nucleic acid amplification technology
探针导向重组酶介导等温扩增法检测TB_rpoB基因突变的应用价值
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[Abstract] Objective: To establish a probe-directed recombinase amplification method (PDRA) capable of rapidly detecting mutations at position 516 of the MTB rpoB gene. Methods: Four probes were designed— one wild-type probe (P-W) and three mutant probes (P-GGC, P-GTC, P-TAC)—along with a common downstream primer. Four detection systems were constructed accordingly: TB-516-W, TB-516-GGC, TB-516-GTC, and TB-516-TAC. These systems underwent isothermal amplification at 39°C for 40 minutes. The presence and type of mutation at MTB rpoB gene position 516 were determined by assessing the positive control threshold time. The sensitivity of each detection system was evaluated using the same probe to detect its corresponding recombinant plasmid. Meanwhile, the specificity of the system was assessed by testing a single recombinant plasmid with different probes, based on the positive threshold time. Finally, the established PDRA method was applied to detect rifampicin-resistant MTB strains in six clinical isolates and rifampicin-positive sputum samples from 35 patients, with results compared against those obtained by first-generation sequencing. Results: All four detection systems demonstrated 100% sensitivity when testing their respective recombinant plasmids. Moreover, the specificity of each system was excellent, as evidenced by early positive threshold times when probes perfectly matched their target sequences, and delayed thresholds when mismatches occurred. Notably, stable differences in positive threshold times were observed among the four systems: TB-516-W, TB-516-GGC, TB-516-GTC, and TB-516-TAC, with threshold time differences of 7.0, 5.8, 10.0, and 7.0 minutes, respectively. The PDRA results for the six rifampicin-resistant MTB strains and 35 rifampicin-positive sputum samples were fully consistent with the sequencing data. Conclusion: This study has preliminarily developed a PDRA method suitable for detecting mutations at MTB rifampicin-resistant rpoB gene position 516. The method exhibits high sensitivity, excellent specificity, and holds significant potential for laboratory applications.
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