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Multiplex LNA probe-based RAP assay for rapid and highly sensitive detection of rifampicin-resistant Mycobacterium tuberculosis
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 sensitivit...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2023
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10172479/ https://www.ncbi.nlm.nih.gov/pubmed/37180280 http://dx.doi.org/10.3389/fmicb.2023.1141424 |
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author | Zhang, Ruiqing Ou, Xichao Sun, Xiuli Fan, Guohao Zhao, Bing Tian, Fengyu Li, Fengyu Shen, Xinxin Zhao, Yanlin Ma, Xuejun |
author_facet | Zhang, Ruiqing Ou, Xichao Sun, Xiuli Fan, Guohao Zhao, Bing Tian, Fengyu Li, Fengyu Shen, Xinxin Zhao, Yanlin Ma, Xuejun |
author_sort | Zhang, Ruiqing |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-10172479 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-101724792023-05-12 Multiplex LNA probe-based RAP assay for rapid and highly sensitive detection of rifampicin-resistant Mycobacterium tuberculosis Zhang, Ruiqing Ou, Xichao Sun, Xiuli Fan, Guohao Zhao, Bing Tian, Fengyu Li, Fengyu Shen, Xinxin Zhao, Yanlin Ma, Xuejun Front Microbiol Microbiology 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. Frontiers Media S.A. 2023-04-27 /pmc/articles/PMC10172479/ /pubmed/37180280 http://dx.doi.org/10.3389/fmicb.2023.1141424 Text en Copyright © 2023 Zhang, Ou, Sun, Fan, Zhao, Tian, Li, Shen, Zhao and Ma. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Microbiology Zhang, Ruiqing Ou, Xichao Sun, Xiuli Fan, Guohao Zhao, Bing Tian, Fengyu Li, Fengyu Shen, Xinxin Zhao, Yanlin Ma, Xuejun Multiplex LNA probe-based RAP assay for rapid and highly sensitive detection of rifampicin-resistant Mycobacterium tuberculosis |
title | Multiplex LNA probe-based RAP assay for rapid and highly sensitive detection of rifampicin-resistant Mycobacterium tuberculosis |
title_full | Multiplex LNA probe-based RAP assay for rapid and highly sensitive detection of rifampicin-resistant Mycobacterium tuberculosis |
title_fullStr | Multiplex LNA probe-based RAP assay for rapid and highly sensitive detection of rifampicin-resistant Mycobacterium tuberculosis |
title_full_unstemmed | Multiplex LNA probe-based RAP assay for rapid and highly sensitive detection of rifampicin-resistant Mycobacterium tuberculosis |
title_short | Multiplex LNA probe-based RAP assay for rapid and highly sensitive detection of rifampicin-resistant Mycobacterium tuberculosis |
title_sort | multiplex lna probe-based rap assay for rapid and highly sensitive detection of rifampicin-resistant mycobacterium tuberculosis |
topic | Microbiology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10172479/ https://www.ncbi.nlm.nih.gov/pubmed/37180280 http://dx.doi.org/10.3389/fmicb.2023.1141424 |
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