{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T23:35:11Z","timestamp":1792798511442,"version":"4.5.0"},"reference-count":50,"publisher":"American Society for Microbiology","issue":"7","license":[{"start":{"date-parts":[[2025,7,1]],"date-time":"2025-07-01T00:00:00Z","timestamp":1751328000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2025,7,1]],"date-time":"2025-07-01T00:00:00Z","timestamp":1751328000000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"name":"Department of Science and Technology of Guangdong Province, P.R.China","award":["2022B1111020003"],"award-info":[{"award-number":["2022B1111020003"]}],"id":[{"id":"https:\/\/ror.org\/00tjzgn92","id-type":"ROR","asserted-by":"crossref"}]},{"DOI":"10.13039\/100017695","name":"Health Commission of Hunan Province","doi-asserted-by":"publisher","award":["D202303086921"],"award-info":[{"award-number":["D202303086921"]}],"id":[{"id":"10.13039\/100017695","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"published-print":{"date-parts":[[2025,7]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Accurate and rapid differentiation of\n                      <jats:italic toggle=\"yes\">Mycobacterium tuberculosis<\/jats:italic>\n                      (\n                      <jats:italic toggle=\"yes\">M. tuberculosis<\/jats:italic>\n                      ) and nontuberculous mycobacteria (NTM) remains challenging, especially in smear-negative samples. Routine diagnostic methods often lack specificity and rapidity, highlighting the urgency of developing applicative molecular diagnostics to guide clinical management and improve patient outcomes. We developed and validated a multiplex PCR assay employing TaqMan probes targeting the\n                      <jats:italic toggle=\"yes\">IS6110<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">rpoB<\/jats:italic>\n                      genes to simultaneously detect\n                      <jats:italic toggle=\"yes\">M. tuberculosis<\/jats:italic>\n                      and 23 NTM species in a single reaction, while avoiding cross-reactivity. Analytical performance was assessed using reference strains, and clinical feasibility was evaluated in a prospective, single-center pilot study involving 96 smear-negative presumptive tuberculosis patients. The assay\u2019s diagnostic performance was compared to mycobacterial culture, DNA sequencing, and the Xpert MTB\/RIF assay. The multiplex PCR assay demonstrated a wide linear dynamic range (10\n                      <jats:sup>8<\/jats:sup>\n                      \u201310\n                      <jats:sup>3<\/jats:sup>\n                      copies\/mL), low limit of detection (10\n                      <jats:sup>3<\/jats:sup>\n                      copies\/mL), high precision (intra-assay CV 0.05\u20131.18%, inter-assay CV 0.08\u20132.57%), and complete specificity for both\n                      <jats:italic toggle=\"yes\">M. tuberculosis<\/jats:italic>\n                      and NTM. In the clinical feasibility study, using a combination of clinical diagnosis and mycobacterial culture as reference standards, it achieved sensitivities of 73.33% (\n                      <jats:italic toggle=\"yes\">M. tuberculosis<\/jats:italic>\n                      ) and 100% (NTM), and specificities of 100% (\n                      <jats:italic toggle=\"yes\">M. tuberculosis<\/jats:italic>\n                      ) and 94.33% (NTM). Notably, it reliably identified\n                      <jats:italic toggle=\"yes\">M. tuberculosis<\/jats:italic>\n                      \/NTM co-infections. The Multiplex PCR assay fills a critical gap in the rapid, accurate diagnosis of\n                      <jats:italic toggle=\"yes\">M. tuberculosis<\/jats:italic>\n                      and NTM in challenging clinical specimens. With a cost of $5 per test and a turnaround time of 3 h, our assay has the potential to improve patient care and optimize mycobacterial infection treatment strategies in resource-limited settings.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Rapid and accurate differentiation between\n                        <jats:italic toggle=\"yes\">Mycobacterium tuberculosis<\/jats:italic>\n                        (\n                        <jats:italic toggle=\"yes\">M. tuberculosis<\/jats:italic>\n                        ) and nontuberculous mycobacteria (NTM) is essential for ensuring timely and appropriate treatment, especially in high tuberculosis (TB) burden regions. Conventional diagnostic methods, such as smear microscopy and culture, often lack the sensitivity or speed needed for reliable results in smear-negative cases, risking misdiagnosis and delayed care. In this study, we developed a novel multiplex real-time PCR assay capable of simultaneously detecting\n                        <jats:italic toggle=\"yes\">M. tuberculosis<\/jats:italic>\n                        and up to 23 clinically relevant NTM species with high specificity and sensitivity. By targeting distinct genetic markers for\n                        <jats:italic toggle=\"yes\">M. tuberculosis<\/jats:italic>\n                        and NTM, our assay provides a cost-effective, 3-h diagnostic solution that enhances diagnostic accuracy in challenging samples. This innovation addresses a critical gap in mycobacterial diagnostics, supporting improved patient outcomes and aligning with global health priorities for the control and elimination of TB.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.02316-24","type":"journal-article","created":{"date-parts":[[2025,5,30]],"date-time":"2025-05-30T09:00:21Z","timestamp":1748595621000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":7,"title":["Simultaneous detection and differentiation of\n                    <i>Mycobacterium tuberculosis<\/i>\n                    and nontuberculous mycobacteria in smear-negative sputum by a multiplex PCR assay: a clinical feasibility 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