{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,10]],"date-time":"2026-09-10T09:09:06Z","timestamp":1789031346543,"version":"build-2803163510"},"reference-count":29,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,9,10]],"date-time":"2026-09-10T00:00:00Z","timestamp":1788998400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100017170","name":"Thailand Science Research and Innovation","doi-asserted-by":"publisher","award":["FRB680102\/0162"],"award-info":[{"award-number":["FRB680102\/0162"]}],"id":[{"id":"10.13039\/501100017170","id-type":"DOI","asserted-by":"publisher"}]},{"award":["FRB680102\/0162"],"award-info":[{"award-number":["FRB680102\/0162"]}],"id":[{"id":"https:\/\/ror.org\/000025p04","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100002842","name":"Chiang Mai University","doi-asserted-by":"publisher","award":["207555"],"award-info":[{"award-number":["207555"]}],"id":[{"id":"10.13039\/501100002842","id-type":"DOI","asserted-by":"publisher"}]},{"award":["207555"],"award-info":[{"award-number":["207555"]}],"id":[{"id":"https:\/\/ror.org\/05m2fqn25","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biosensors"],"abstract":"<jats:p>Drug-resistant tuberculosis remains a major challenge to tuberculosis control, particularly in settings with limited access to conventional molecular testing. This study developed and preliminarily evaluated RR-TB GoldDx and Hr-TB GoldDx, rapid visual assays for detecting rifampicin- and isoniazid-resistance-associated mutations in Mycobacterium tuberculosis. The assays combine recombinase polymerase amplification with unmodified gold nanoparticles (AuNPs) for amplification confirmation and thiol-probe-modified AuNPs for detecting resistance-associated mutations at rpoB codons 516, 526, and 531, katG codon 315, and position \u221215 of the fabG1\u2013inhA promoter. Evaluation using DNA from 50 M. tuberculosis isolates yielded positive internal-control results for all samples. RR-TB GoldDx achieved accuracies of 84% and 86% against phenotypic drug-susceptibility testing and DNA sequencing, respectively, with Cohen\u2019s kappa values of 0.68 and 0.72. Hr-TB GoldDx achieved 80% accuracy and a kappa value of 0.60 against phenotypic testing, while the inhA promoter and katG assays achieved sequencing-based accuracies of 84% and 86%, respectively. Results were available within 1 h at an estimated reagent cost of THB 500\u2013550 (approximately USD 15\u201316) per test without conventional thermocycling, real-time fluorescence detection, or gel electrophoresis. These instrument-minimal assays show potential for rapid drug-resistance screening but require validation in larger, genetically diverse cohorts and direct respiratory specimens.<\/jats:p>","DOI":"10.3390\/bios16090506","type":"journal-article","created":{"date-parts":[[2026,9,10]],"date-time":"2026-09-10T08:07:41Z","timestamp":1789027661000},"page":"506","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Development and Preliminary Evaluation of RR-TB GoldDx and Hr-TB GoldDx: Rapid Visual RPA-Gold Nanoparticle Assays for Detecting Rifampicin- and Isoniazid-Resistance-Associated\u00a0Mutations in Mycobacterium tuberculosis"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6648-8972","authenticated-orcid":false,"given":"Usanee","family":"Wattananandkul","sequence":"first","affiliation":[{"name":"Division\u00a0of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand"},{"name":"Infectious Diseases Research Unit (IDRU), Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0829-3413","authenticated-orcid":false,"given":"Sukanya","family":"Saikaew","sequence":"additional","affiliation":[{"name":"Division\u00a0of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand"},{"name":"Office of Research Administration, Chiang Mai University, Chiang Mai 50200, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4987-5505","authenticated-orcid":false,"given":"Sirikwan","family":"Sangboonruang","sequence":"additional","affiliation":[{"name":"Division\u00a0of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand"},{"name":"Infectious Diseases Research Unit (IDRU), Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rodjana","family":"Pongsararuk","sequence":"additional","affiliation":[{"name":"Office of Disease Prevention and Control Region 1 (ODPC-1) Chiang Mai, Department of Disease Control, The Ministry of Public Health, Muang District, Chiang Mai 50100, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4882-516X","authenticated-orcid":false,"given":"Prapaporn","family":"Srilohasin","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand"},{"name":"Drug Resistant Tuberculosis Research (DR-TB) Fund, Siriraj Foundation, Bangkok 10700, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8560-6540","authenticated-orcid":false,"given":"Bordin","family":"Butr-Indr","sequence":"additional","affiliation":[{"name":"Division\u00a0of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand"},{"name":"Infectious Diseases Research Unit (IDRU), Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7137-4750","authenticated-orcid":false,"given":"Sorasak","family":"Intorasoot","sequence":"additional","affiliation":[{"name":"Division\u00a0of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand"},{"name":"Infectious Diseases Research Unit (IDRU), Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1441-9641","authenticated-orcid":false,"given":"Chayada Sitthidet","family":"Tharinjaroen","sequence":"additional","affiliation":[{"name":"Division\u00a0of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand"},{"name":"Infectious Diseases Research Unit (IDRU), Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2078-3201","authenticated-orcid":false,"given":"Natthawat","family":"Semakul","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Faculty of Sciences, Chiang Mai University, Chiang Mai 50200, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Surachet","family":"Arunothong","sequence":"additional","affiliation":[{"name":"Office of Disease Prevention and Control Region 1 (ODPC-1) Chiang Mai, Department of Disease Control, The Ministry of Public Health, Muang District, Chiang Mai 50100, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Angkana","family":"Chaiprasert","sequence":"additional","affiliation":[{"name":"Drug Resistant Tuberculosis Research (DR-TB) Fund, Siriraj Foundation, Bangkok 10700, Thailand"},{"name":"Office for Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8812-5139","authenticated-orcid":false,"given":"Khajornsak","family":"Tragoolpua","sequence":"additional","affiliation":[{"name":"Division\u00a0of Clinical Microbiology, Department of Medical Technology, Faculty of 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Global Tuberculosis Report 2025, World Health Organization. Available online: https:\/\/www.who.int\/publications\/i\/item\/9789240116924."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"617","DOI":"10.1038\/s41579-024-01025-1","article-title":"Drug-resistant tuberculosis: A persistent global health concern","volume":"22","author":"Farhat","year":"2024","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"22","DOI":"10.1038\/s41572-024-00504-2","article-title":"Multidrug-resistant tuberculosis","volume":"10","author":"Dheda","year":"2024","journal-title":"Nat. Rev. Dis. Primers"},{"key":"ref_4","unstructured":"World Health Organization (2025). WHO Consolidated Guidelines on Tuberculosis: Module 3: Diagnosis, World Health Organization. Available online: https:\/\/www.who.int\/publications\/i\/item\/9789240107984."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2371","DOI":"10.1007\/s15010-024-02295-w","article-title":"Accurate and Affordable Detection of Rifampicin and Isoniazid Resistance in Tuberculosis Sputum Specimens by Multiplex PCR-Multiple Probes Melting Analysis","volume":"52","author":"Xie","year":"2024","journal-title":"Infection"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"3375","DOI":"10.2147\/IDR.S263082","article-title":"Genotypic distribution and a potential diagnostic assay of multidrug-resistant tuberculosis in northern Thailand","volume":"13","author":"Anukool","year":"2020","journal-title":"Infect. Drug Resist."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Thant, Y.M., Saikaew, S., Tharinjaroen, C.S., Phunpae, P., Pongsararuk, R., Preechasuth, K., Butr-Indr, B., Intorasoot, S., Tragoolpua, K., and Chaiprasert, A. (2022). Combined Locked Nucleic Acid Probes and High-Resolution Melting Curve Analysis for Detection of Rifampicin-Resistant Tuberculosis in Northern Thailand. Diagnostics, 12.","DOI":"10.3390\/diagnostics12102307"},{"key":"ref_8","unstructured":"World Health Organization (2023). Catalogue of Mutations in Mycobacterium tuberculosis Complex and Their Association with Drug Resistance, World Health Organization. [2nd ed.]. Available online: https:\/\/www.who.int\/publications\/i\/item\/9789240082410."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"167","DOI":"10.1016\/j.cmi.2016.09.006","article-title":"Consensus numbering system for the rifampicin resistance-associated rpoB gene mutations in pathogenic mycobacteria","volume":"23","author":"Andre","year":"2017","journal-title":"Clin. Microbiol. Infect."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"e02091-20","DOI":"10.1128\/AAC.02091-20","article-title":"Systematic review of mutations associated with isoniazid resistance points to continuing evolution and subsequent evasion of molecular detection, and potential for emergence of multidrug resistance in clinical strains of Mycobacterium tuberculosis","volume":"65","author":"Valafar","year":"2021","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_11","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":"Lobato","year":"2018","journal-title":"TrAC Trends Anal. Chem."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"947","DOI":"10.1373\/clinchem.2015.245829","article-title":"Recombinase polymerase amplification for diagnostic applications","volume":"62","author":"Daher","year":"2016","journal-title":"Clin. Chem."},{"key":"ref_13","first-page":"616","article-title":"Evaluation of Multidrug Resistant Loop-mediated Isothermal Amplification Assay for Detecting the Drug Resistance of Mycobacterium tuberculosis","volume":"34","author":"Liu","year":"2021","journal-title":"Biomed. Environ. Sci."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Singpanomchai, N., Akeda, Y., Tomono, K., Tamaru, A., Santanirand, P., and Ratthawongjirakul, P. (2021). Rapid detection of multidrug-resistant tuberculosis based on allele-specific recombinase polymerase amplification and colorimetric detection. PLoS ONE, 16.","DOI":"10.1371\/journal.pone.0253235"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Ferrari, E. (2023). Gold Nanoparticle-Based Plasmonic Biosensors. Biosensors, 13.","DOI":"10.3390\/bios13030411"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Enea, M., Leite, A., Franco, R., and Pereira, E. (2024). Gold Nanoprobes for Robust Colorimetric Detection of Nucleic Acid Sequences Related to Disease Diagnostics. Nanomaterials, 14.","DOI":"10.3390\/nano14221833"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"4464","DOI":"10.1021\/la204289k","article-title":"Colloidal stability of gold nanoparticles modified with thiol compounds: Bio-conjugation and application in cancer cell imaging","volume":"28","author":"Gao","year":"2012","journal-title":"Langmuir"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"14036","DOI":"10.1073\/pnas.0406115101","article-title":"Colorimetric detection of DNA sequences based on electrostatic interactions with unmodified gold nanoparticles","volume":"101","author":"Li","year":"2004","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_19","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":"Veigas","year":"2013","journal-title":"J. Nanobiotechnology"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"4348","DOI":"10.1038\/ncomms5348","article-title":"Quantifying thiol\u2013gold interactions towards the efficient strength control","volume":"5","author":"Xue","year":"2014","journal-title":"Nat. Commun."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Saikaew, S., Sangboonruang, S., Pongsararuk, R., Srilohasin, P., Butr-Indr, B., Intorasoot, S., Phunpae, P., Tharinjaroen, C.S., Arunothong, S., and Panyasit, W. (2025). Gold Nanoparticle-Enhanced Recombinase Polymerase Amplification for Rapid Visual Detection of Mycobacterium tuberculosis. Biosensors, 15.","DOI":"10.3390\/bios15090607"},{"key":"ref_22","unstructured":"MedCalc Software Ltd. (2026, July 01). Diagnostic Test Evaluation. Available online: https:\/\/www.medcalc.org\/calc\/diagnostic_test.php."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"159","DOI":"10.2307\/2529310","article-title":"The measurement of observer agreement for categorical data","volume":"33","author":"Landis","year":"1977","journal-title":"Biometrics"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Son, H. (2024). Harnessing CRISPR\/Cas Systems for DNA and RNA Detection: Principles, Techniques, and Challenges. Biosensors, 14.","DOI":"10.3390\/bios14100460"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"e00610-21","DOI":"10.1128\/Spectrum.00610-21","article-title":"A rapid drug resistance genotyping workflow for Mycobacterium tuberculosis, using targeted isothermal amplification and nanopore sequencing","volume":"9","author":"Gliddon","year":"2021","journal-title":"Microbiol. Spectr."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"813","DOI":"10.1007\/s00216-018-1503-y","article-title":"Adenoviral detection by recombinase polymerase amplification and vertical flow paper microarray","volume":"411","author":"Nybond","year":"2019","journal-title":"Anal. Bioanal. Chem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"4126","DOI":"10.1021\/acs.analchem.1c00013","article-title":"Reverse transcription recombinase polymerase amplification coupled with CRISPR-Cas12a for facile and highly sensitive colorimetric SARS-CoV-2 detection","volume":"93","author":"Zhang","year":"2021","journal-title":"Anal. Chem."},{"key":"ref_28","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 strains from Thailand and development of a multiplex allele-specific PCR assay for mutation detection","volume":"14","author":"Prammananan","year":"2008","journal-title":"Clin. Microbiol. Infect."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"276","DOI":"10.1016\/j.diagmicrobio.2017.08.016","article-title":"Laboratory evaluation of the Anyplex II MTB\/MDR and MTB\/XDR tests based on multiplex real-time PCR and melting-temperature analysis to identify Mycobacterium tuberculosis and drug resistance","volume":"89","author":"Igarashi","year":"2017","journal-title":"Diagn. Microbiol. Infect. Dis."}],"container-title":["Biosensors"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-6374\/16\/9\/506\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,10]],"date-time":"2026-09-10T08:36:10Z","timestamp":1789029370000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-6374\/16\/9\/506"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,10]]},"references-count":29,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,9]]}},"alternative-id":["bios16090506"],"URL":"https:\/\/doi.org\/10.3390\/bios16090506","relation":{},"ISSN":["2079-6374"],"issn-type":[{"value":"2079-6374","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,10]]}}}