ArticleFrontiers in microbiology2023
Multiplex LNA probe-based RAP assay for rapid and highly sensitive detection of rifampicin-resistant
Article in Frontiers in microbiology, 2023. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 6 papers.
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6 citing papers in PubMed, 6 citations in OpenAlex.
- Application of Universal LNA Probe-Mediated Multiplex Real-Time PCR for the Detection of Antibiotic Resistance Genes.Molecular biotechnology · 2026Article
- Molecular epidemiology of drug resistance and transmission ofMicrobiology spectrum · 2026Article
- Review
- A duplex one-step recombinase aided PCR assay for the rapid and sensitive detection of the isoniazid resistance genes katG and inhA inFrontiers in microbiology · 2025Article
- A Multiplex Recombinase-Aided qPCR Assay for Highly Sensitive and Rapid Detection of khe, blaJournal of clinical laboratory analysis · 2024Article
- Partial Characterization of Three Bacteriophages Isolated from Aquaculture Hatchery Water and Their Potential in the Biocontrol ofMicroorganisms · 2024Article
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Authors and funding
10 authors at 5 institutions in 1 country.
Funding
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Abstract
Objectives: The World Health Organization (WHO) Global tuberculosis Report 2021 stated that rifampicin-resistant tuberculosis (RR-TB) remains a major public health threat. However, the in-practice diagnostic techniques for RR-TB have a variety of limitations including longer time, lack of sensitivity, and undetectable low proportion of heterogeneous drug resistance. Methods: Here we developed a multiplex LNA probe-based RAP method (MLP-RAP) for more sensitive detection of multiple point mutations of the RR-TB and its heteroresistance. A total of 126 clinical isolates and 78 sputum samples collected from the National Tuberculosis Reference Laboratory, China CDC, were tested by MLP-RAP assay. In parallel, qPCR and Sanger sequencing of nested PCR product assay were also performed for comparison. Results: The sensitivity of the MLP-RAP assay could reach 5 copies/μl using recombinant plasmids, which is 20 times more sensitive than qPCR (100 copies/μl). In addition, the detection ability of rifampicin heteroresistance was 5%. The MLP-RAP assay had low requirements (boiling method) for nucleic acid extraction and the reaction could be completed within 1 h when placed in a fluorescent qPCR instrument. The result of the clinical evaluation showed that the MLP-RAP method could cover codons 516, 526, 531, and 533 with good specificity. 41 out of 78 boiled sputum samples were detected positive by MLP-RAP assay, which was further confirmed by Sanger sequencing of nested PCR product assay, on the contrary, qPCR was able to detect 32 samples only. Compared with Sanger sequencing of nested PCR product assay, both the specificity and sensitivity of the MLP-RAP assay were 100%. Conclusion: MLP-RAP assay can detect RR-TB infection with high sensitivity and specificity, indicating that this assay has the prospect of being applied for rapid and sensitive RR-TB detection in general laboratories where fluorescent qPCR instrument is available.
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