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Focusing on the research and development and application of isothermal nucleic acid amplification technology

Application Value of Probe-Guided Recombinase-Mediated Isothermal Amplification for Detecting Mutations in the TB_rpoB Gene—CDC

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【Abstract】Objective: To establish a probe-directed recombinase amplification (PDRA) method for the rapid detection of mutations at position 516 of the MTB rpoB gene. Methods: Four probes were designed—1 wild-type probe (P-W) and 3 mutant probes (P-GGC, P-GTC, and P-TAC)—along with a common downstream primer. Four detection systems were constructed: TB-516-W, TB-516-GGC, TB-516-GTC, and TB-516-TAC. These systems underwent isothermal amplification at a constant temperature of 39°C for 40 minutes. The presence and type of mutation at position 516 of the MTBrpoB gene were determined by analyzing the positive threshold time. The sensitivity of each detection system was evaluated by using the same probe to detect corresponding recombinant plasmids. The specificity of each detection system was assessed by using different probes to detect a single recombinant plasmid and determining the positive threshold time for each probe. The established PDRA method was applied to detect 6 rifampicin-resistant MTB strains and 35 sputum samples positive for MTB culture, and the results were compared with those obtained by first-generation sequencing. Results: The sensitivity of the four detection systems for detecting their corresponding recombinant plasmids was 1,000 copies/μL. All four detection systems exhibited good specificity; when the probe perfectly matched the target sequence, the positive threshold time was shorter, whereas when the probe did not fully match the target sequence, the positive threshold time was longer. A stable difference in positive threshold times was observed among the systems: the differences between the positive threshold times for the TB-516-W, TB-516-GGC, TB-516-GTC, and TB-516-TAC systems were 7.0, 5.8, 10.0, and 7.0 minutes, respectively. The results obtained by PDRA for the 6 rifampicin-resistant MTB strains and 35 MTB-positive sputum samples were consistent with the sequencing results. Conclusion: This study has preliminarily established a PDRA method that can be used for the detection of mutations at position 516 of the MTB rpoB gene associated with rifampicin resistance. The method demonstrates high sensitivity and good specificity, making it valuable for laboratory applications.

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Study on the Development of a Detection Method for Non-Tuberculous Mycobacteria Based on Recombinase-Aided Isothermal Amplification—Zhejiang Inspection and Quarantine & Hangzhou Center for Disease Control and Prevention

The Combined Diagnostic Value of RNA Isothermal Amplification Real-Time Detection Technology and Fluorescent Quantitative PCR for Rapid Diagnosis of Pulmonary Alveolar Lavage Fluid in Patients with Sputum-Negative Tuberculosis—Wang Jing

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Study on the Development of a Detection Method for Non-Tuberculous Mycobacteria Based on Recombinase-Aided Isothermal Amplification—Zhejiang Inspection and Quarantine & Hangzhou Center for Disease Control and Prevention

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The Combined Diagnostic Value of RNA Isothermal Amplification Real-Time Detection Technology and Fluorescent Quantitative PCR for Rapid Diagnosis of Pulmonary Alveolar Lavage Fluid in Patients with Sputum-Negative Tuberculosis—Wang Jing