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We first constructed a reference plasmid library comprising 40 prevalent mutations in the target genes\u2019 resistance determining regions and promoters, serving as positive controls. Our assay utilizes a four-tube asymmetric PCR method with specifically designed molecular beacon probes, enabling simultaneous detection of all 40 mutations. We evaluated the assay\u2019s effectiveness using DNA isolated from 50 clinically confirmed <jats:italic>M. tuberculosis<\/jats:italic> sputum specimens, comparing our results with those obtained from Sanger sequencing and retrospective validation involving bacteriological culture and phenotypic drug susceptibility testing (pDST). We also included the commercial Xpert MTB\/RIF assay for accuracy comparison.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Results<\/jats:title>\n                <jats:p>Our data demonstrated remarkable sensitivity in detecting resistance to RIF and INH, achieving values of 93.33% and 95.24%, respectively, with a specificity of 100%. The concordance between our assay and pDST was 98.00%. Furthermore, the accuracy of our assay was comparable to both Sanger sequencing and the Xpert assay. Importantly, our assay boasts a 4.2-h turnaround time and costs only $10 per test, making it an optimal choice for peripheral healthcare settings.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Conclusion<\/jats:title>\n                <jats:p>These findings highlight our assay\u2019s potential as a promising tool for rapidly, accurately, and affordably detecting MDR-TB.<\/jats:p>\n              <\/jats:sec>","DOI":"10.1007\/s15010-024-02295-w","type":"journal-article","created":{"date-parts":[[2024,6,17]],"date-time":"2024-06-17T09:02:14Z","timestamp":1718614934000},"page":"2371-2398","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":11,"title":["Accurate and affordable detection of rifampicin and isoniazid resistance in Tuberculosis sputum specimens by multiplex PCR-multiple probes melting analysis"],"prefix":"10.1007","volume":"52","author":[{"given":"Long","family":"Xie","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiao-Ya","family":"Zhu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Li","family":"Xu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiao-Xie","family":"Xu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ze-Fan","family":"Ruan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ming-Xiang","family":"Huang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Li","family":"Chen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xi-Wen","family":"Jiang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2024,6,17]]},"reference":[{"key":"2295_CR1","volume-title":"Global tuberculosis report 2021","author":"World Health O","year":"2021","unstructured":"World Health O. 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This approval was in line with the Helsinki declaration as revised in 2013 and its later amendments. All participants provided written informed consent to the collection, the storage and the use of biological samples for scientific analysis before participating in the study. To ensure ethical compliance, data pertinent to disease control were collected and subsequently anonymized for analysis. All experimental protocols of this study were performed following national technical specification for TB prevention and control.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethical approval, consent to participate and biosafety statement"}}]}}