{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,10]],"date-time":"2026-09-10T19:23:30Z","timestamp":1789068210920,"version":"build-2803163510"},"reference-count":35,"publisher":"Public Library of Science (PLoS)","issue":"6","license":[{"start":{"date-parts":[[2021,6,11]],"date-time":"2021-06-11T00:00:00Z","timestamp":1623369600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100002873","name":"Chulalongkorn University","doi-asserted-by":"publisher","award":["NRU-59-012-HR"],"award-info":[{"award-number":["NRU-59-012-HR"]}],"id":[{"id":"10.13039\/501100002873","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>Multidrug-resistant tuberculosis (MDR-TB) poses a serious threat to TB control. Early diagnosis and proper treatment are essential factors to limit the spread of the disease. The existing molecular tests for MDR-TB usually require specific instruments, steady power supply, and routine maintenance, which might be obstacles for low-resource settings. This study aimed to develop allele-specific isothermal recombinase polymerase amplification (allele-specific RPA) to simultaneously detect the most common mutations in the<jats:italic>rpoB<\/jats:italic>gene at codons 516, 526, and 531, which are associated with rifampicin resistance, and in the<jats:italic>katG<\/jats:italic>gene at codon 315, which is related to isoniazid resistance. Allele-specific primers targeting four major mutations,<jats:italic>rpoB<\/jats:italic>516,<jats:italic>rpoB<\/jats:italic>526,<jats:italic>rpoB<\/jats:italic>531, and<jats:italic>katG<\/jats:italic>315, were constructed and used in individual RPA reactions. The RPA amplicons were endpoints detected by the naked eye immediately after applying SYBR Green I. The optimised RPA assay was evaluated with the<jats:italic>Mycobacterium tuberculosis<\/jats:italic>wild-type strain H37Rv and 141 clinical<jats:italic>M<\/jats:italic>.<jats:italic>tuberculosis<\/jats:italic>isolates. The results revealed that allele-specific RPA combined with SYBR Green I detection (AS-RPA\/SYBR) detected these four major mutations with 100% sensitivity and specificity relative to DNA sequencing. The limits of detection for these particular mutations with AS-RPA\/SYBR were 5 ng. As a result of the outstanding performance of AS-RPA\/SYBR, including its easy setup, speed, lack of a specific instrument requirement, and lack of cross-reaction with other bacteria, this technique may be integrated for the molecular diagnosis of MDR-TB, especially in low-resource settings.<\/jats:p>","DOI":"10.1371\/journal.pone.0253235","type":"journal-article","created":{"date-parts":[[2021,6,11]],"date-time":"2021-06-11T17:37:09Z","timestamp":1623433029000},"page":"e0253235","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":37,"title":["Rapid detection of multidrug-resistant tuberculosis based on allele-specific recombinase polymerase amplification and colorimetric detection"],"prefix":"10.1371","volume":"16","author":[{"given":"Nuntita","family":"Singpanomchai","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yukihiro","family":"Akeda","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kazunori","family":"Tomono","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aki","family":"Tamaru","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pitak","family":"Santanirand","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3084-0863","authenticated-orcid":true,"given":"Panan","family":"Ratthawongjirakul","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2021,6,11]]},"reference":[{"key":"pone.0253235.ref001","unstructured":"Global tuberculosis report 2020. World Health Oraganization. 2020. Available from: https:\/\/apps.who.int\/iris\/bitstream\/handle\/10665\/336069\/9789240013131-eng.pdf."},{"issue":"1","key":"pone.0253235.ref002","doi-asserted-by":"crossref","first-page":"23","DOI":"10.33073\/pjm-2012-003","article-title":"Characterization of mutations causing rifampicin and isoniazid resistance of Mycobacterium tuberculosis in Syria","volume":"61","author":"A Madania","year":"2012","journal-title":"Pol J Microbiol"},{"issue":"5","key":"pone.0253235.ref003","doi-asserted-by":"crossref","first-page":"446","DOI":"10.1111\/j.1469-0691.2008.01951.x","article-title":"Distribution of rpoB mutations among multidrug-resistant Mycobacterium tuberculosis (MDRTB) strains from Thailand and development of a rapid method for mutation detection","volume":"14","author":"T Prammananan","year":"2008","journal-title":"Clin Microbiol Infect"},{"issue":"5","key":"pone.0253235.ref004","doi-asserted-by":"crossref","first-page":"2209","DOI":"10.1128\/JCM.41.5.2209-2212.2003","article-title":"Mutations in the rpoB gene of multidrug-resistant Mycobacterium tuberculosis isolates from China","volume":"41","author":"J Yue","year":"2003","journal-title":"J Clin Microbiol"},{"issue":"4","key":"pone.0253235.ref005","doi-asserted-by":"crossref","first-page":"396","DOI":"10.1111\/j.1469-0691.2009.02838.x","article-title":"Genotypic analysis of genes associated with isoniazid and ethionamide resistance in MDR-TB isolates from Thailand","volume":"16","author":"S Boonaiam","year":"2010","journal-title":"Clin Microbiol Infect"},{"issue":"Suppl 4","key":"pone.0253235.ref006","doi-asserted-by":"crossref","first-page":"539","DOI":"10.1007\/s00586-012-2334-8","article-title":"Factors that influence current tuberculosis epidemiology","volume":"22","author":"J-P Millet","year":"2013","journal-title":"Eur Spine J"},{"issue":"5","key":"pone.0253235.ref007","doi-asserted-by":"crossref","first-page":"1484","DOI":"10.1128\/JCM.02289-08","article-title":"Potential of rapid diagnosis for controlling drug-susceptible and drug-resistant tuberculosis in communities where Mycobacterium tuberculosis infections are highly prevalent","volume":"47","author":"PW Uys","year":"2009","journal-title":"J Clin Microbiol"},{"issue":"2","key":"pone.0253235.ref008","doi-asserted-by":"crossref","first-page":"64","DOI":"10.4046\/trd.2015.78.2.64","article-title":"Diagnosis of pulmonary tuberculosis: recent advances and diagnostic algorithms","volume":"78","author":"YJ Ryu","year":"2015","journal-title":"Tuberc Respir Dis"},{"issue":"4","key":"pone.0253235.ref009","doi-asserted-by":"crossref","first-page":"1089","DOI":"10.1128\/JCM.02838-15","article-title":"Mycobacterium and aerobic actinomycete Culture: are two medium types and extended incubation times necessary?","volume":"54","author":"PJ Simner","year":"2016","journal-title":"J Clin Microbiol"},{"issue":"3","key":"pone.0253235.ref010","first-page":"457","article-title":"Diagnostic test of sputum genexpert MTB\/RIF for smear negative pulmonary tuberculosis","volume":"47","author":"W Reechaipichitkul","year":"2016","journal-title":"Southeast Asian J Trop Med Public Health"},{"key":"pone.0253235.ref011","unstructured":"WHO policy statement: molecular line probe assays for rapid screening of patients at risk of multidrug-resistant tuberculosis. World Health Oraganization. 2008. Available from: http:\/\/www.who.int\/tb\/features_archive\/expert_group_report_june08.pdf."},{"issue":"7","key":"pone.0253235.ref012","doi-asserted-by":"crossref","first-page":"e204","DOI":"10.1371\/journal.pbio.0040204","article-title":"DNA detection using recombination proteins","volume":"4","author":"O Piepenburg","year":"2006","journal-title":"PLoS Biol"},{"issue":"8","key":"pone.0253235.ref013","doi-asserted-by":"crossref","first-page":"e103091","DOI":"10.1371\/journal.pone.0103091","article-title":"Rapid detection of Mycobacterium tuberculosis by recombinase polymerase amplification","volume":"9","author":"DS Boyle","year":"2014","journal-title":"PLoS One"},{"key":"pone.0253235.ref014","doi-asserted-by":"crossref","first-page":"98","DOI":"10.1016\/j.ab.2017.12.031","article-title":"Automated real-time detection of drug-resistant Mycobacterium tuberculosis on a lab-on-a-disc by recombinase polymerase amplification","volume":"544","author":"ILG Law","year":"2018","journal-title":"Anal Biochem"},{"issue":"17","key":"pone.0253235.ref015","first-page":"9017","article-title":"Isothermal point mutation detection: toward a first-pass screening strategy for multidrug-resistant tuberculosis","volume":"89","author":"BYC Ng","year":"2017","journal-title":"Anal Biochem"},{"issue":"2","key":"pone.0253235.ref016","doi-asserted-by":"crossref","first-page":"e22655","DOI":"10.1002\/jcla.22655","article-title":"Naked eye detection of the Mycobacterium tuberculosis complex by recombinase polymerase amplification\u2014SYBR green I assays","volume":"33","author":"N Singpanomchai","year":"2019","journal-title":"J Clin Lab Anal"},{"issue":"9","key":"pone.0253235.ref017","doi-asserted-by":"crossref","first-page":"e0137791","DOI":"10.1371\/journal.pone.0137791","article-title":"Rapid molecular detection of multidrug-resistant tuberculosis by PCR-nucleic acid lateral flow immunoassay","volume":"10","author":"H Kamphee","year":"2015","journal-title":"PLoS One"},{"key":"pone.0253235.ref018","unstructured":"Cracolici V. Advanced approach: Introduction to the Yaku-Bonczyk primer design method. Michigan State University; 2011."},{"key":"pone.0253235.ref019","unstructured":"The End TB Strategy. World Health Organization. 2014. Available from: http:\/\/www.who.int\/tb\/strategy\/End_TB_Strategy.pdf?ua=1."},{"issue":"1","key":"pone.0253235.ref020","first-page":"1","article-title":"The contribution of molecular biology in diagnosing tuberculosis and detecting antibiotic resistance","volume":"2","author":"C Pierre-Audigier","year":"2013","journal-title":"J Tuberc Res"},{"key":"pone.0253235.ref021","unstructured":"World Health Oraganization. The use of loop-mediated isothermal amplification (TB-LAMP) for the diagnosis of pulmonary tuberculosis: policy guidance: World Health Organization. 2016. Available from: http:\/\/www.who.int\/tb\/publications\/lamp-diagnosis-molecular\/en\/."},{"key":"pone.0253235.ref022","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1155\/2016\/3474396","article-title":"Development of Au-nanoprobes combined with loop-mediated isothermal amplification for detection of isoniazid resistance in Mycobacterium tuberculosis","volume":"2016","author":"J Ckumdee","year":"2016","journal-title":"J Chem"},{"key":"pone.0253235.ref023","doi-asserted-by":"crossref","first-page":"38","DOI":"10.1186\/1477-3155-11-38","article-title":"Isothermal DNA amplification coupled to Au-nanoprobes for detection of mutations associated to Rifampicin resistance in Mycobacterium tuberculosis","volume":"11","author":"B Veigas","year":"2013","journal-title":"J Nanobiotechnology"},{"key":"pone.0253235.ref024","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1016\/j.trac.2017.10.015","article-title":"Recombinase polymerase amplification: basics, applications and recent advances","volume":"98","author":"IM Lobato","year":"2018","journal-title":"Trends Analyt Chem"},{"issue":"7","key":"pone.0253235.ref025","doi-asserted-by":"crossref","first-page":"947","DOI":"10.1373\/clinchem.2015.245829","article-title":"Recombinase polymerase amplification for diagnostic applications","volume":"62","author":"RK Daher","year":"2016","journal-title":"Clin Chem"},{"issue":"44","key":"pone.0253235.ref026","doi-asserted-by":"crossref","DOI":"10.2807\/1560-7917.ES.2015.20.44.30053","article-title":"Development and deployment of a rapid recombinase polymerase amplification Ebola virus detection assay in Guinea in 2015","volume":"20","author":"O Faye","year":"2015","journal-title":"Euro Surveill"},{"issue":"5","key":"pone.0253235.ref027","doi-asserted-by":"crossref","first-page":"792","DOI":"10.3390\/ijms17050792","article-title":"A novel technique to detect EGFR mutations in lung cancer","volume":"17","author":"Y Liu","year":"2016","journal-title":"Int J Mol Sci"},{"key":"pone.0253235.ref028","doi-asserted-by":"crossref","first-page":"446","DOI":"10.1186\/s13071-015-1055-3","article-title":"Isothermal recombinase polymerase amplification (RPA) of Schistosoma haematobium DNA and oligochromatographic lateral flow detection","volume":"8","author":"A Rosser","year":"2015","journal-title":"Parasit Vectors"},{"issue":"8","key":"pone.0253235.ref029","doi-asserted-by":"crossref","first-page":"4413","DOI":"10.1021\/acs.analchem.7b00964","article-title":"Instant, visual, and instrument-free method for on-site screening of GTS 40-3-2 soybean based on body-heat triggered recombinase polymerase amplification","volume":"89","author":"R Wang","year":"2017","journal-title":"Anal Chem"},{"issue":"5","key":"pone.0253235.ref030","doi-asserted-by":"crossref","first-page":"1453","DOI":"10.1007\/s00604-017-2144-0","article-title":"Low-cost genotyping method based on allele-specific recombinase polymerase amplification and colorimetric microarray detection","volume":"184","author":"ES Yamanaka","year":"2017","journal-title":"Microchim Acta"},{"key":"pone.0253235.ref031","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1016\/j.mcp.2009.08.001","article-title":"Enhancing allele-specific PCR for specifically detecting short deletion and insertion DNA mutations","volume":"24","author":"Y Wang","year":"2010","journal-title":"Mol Cell Probes"},{"issue":"3","key":"pone.0253235.ref032","doi-asserted-by":"crossref","first-page":"e0119628","DOI":"10.1371\/journal.pone.0119628","article-title":"Genetic mutations associated with isoniazid resistance in Mycobacterium tuberculosis: a systematic review","volume":"10","author":"M Seifert","year":"2015","journal-title":"PLoS One"},{"key":"pone.0253235.ref033","doi-asserted-by":"crossref","first-page":"3","DOI":"10.25518\/1780-4507.13461","article-title":"Influence of the amplicon length on real-time PCR results","volume":"21","author":"F Debode","year":"2017","journal-title":"Biotechnol Agron Soc Environ"},{"key":"pone.0253235.ref034","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1016\/j.mcp.2017.08.004","article-title":"Rapid and visual detection of Mycobacterium tuberculosis complex using recombinase polymerase amplification combined with lateral flow strips","volume":"36","author":"Q Ma","year":"2017","journal-title":"Mol Cell Probes"},{"issue":"79","key":"pone.0253235.ref035","doi-asserted-by":"crossref","first-page":"49895","DOI":"10.1039\/C7RA09999A","article-title":"Establishment of a rapid and sensitive method based on recombinase polymerase amplification to detect mts90, a new molecular target of Mycobacterium tuberculosis","volume":"7","author":"Y Mo","year":"2017","journal-title":"RSC Adv"}],"container-title":["PLOS ONE"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0253235","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,12,30]],"date-time":"2022-12-30T21:06:44Z","timestamp":1672434404000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0253235"}},"subtitle":[],"editor":[{"given":"Seyed Ehtesham","family":"Hasnain","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2021,6,11]]},"references-count":35,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2021,6,11]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0253235","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,6,11]]}}}