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However, their adoption in clinical laboratories has been limited by challenges such as low detection sensitivity and operational complexity. To address these challenges, we integrated a novel reporter phage into the clinical laboratory workflow for DST of\n                      <jats:italic>Mtb<\/jats:italic>\n                      .\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      A novel\u00a0high-sensitive reporter phage, \u03a6LSN, was constructed by integrating promoter P\n                      <jats:sub>\n                        <jats:italic>left<\/jats:italic>\n                      <\/jats:sub>\n                      , the ribosome-binding site (RBS) of promoter P\n                      <jats:sub>\n                        <jats:italic>smyc<\/jats:italic>\n                      <\/jats:sub>\n                      , and nanoluciferase reporter gene (\n                      <jats:italic>nluc<\/jats:italic>\n                      ) into the genome of the temperature-sensitive phage TM4. Subsequently, a rapid, easy-to-use \u03a6LSN-based workflow was developed for DST of tuberculosis (TB) specimens following mycobacteria growth indicator tube (MGIT) culture, with the distinct advantage of allowing for simultaneous DST of multiple drugs all-in-one plate within 72\u00a0h. The accuracy of the \u03a6LSN DST assay was evaluated against the traditional solid culture assay, using area under the curve (AUC) analysis based on the DeLong test.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      When tested against 62\n                      <jats:italic>Mtb<\/jats:italic>\n                      clinical isolates for susceptibility to first-line anti-tuberculosis drugs (rifampicin, isoniazid, streptomycin, and ethambutol), the \u03a6LSN DST assay showed sensitivities of 93.8%, 96.4%, 100%, and 80%, and specificities of 100%, 97.1%, 100%, and 100%, respectively. Moreover, preliminary prospective validation against 131 positive MGIT cultures demonstrated that the \u03a6LSN DST assay achieved sensitivities of 87.5%, 100%, 100%, and 100%, and specificities of 100%, 99.1%, 99.1%, and 99.1% for the four first-line anti-TB drugs within 72\u00a0h.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>The integration of the BACTEC MGIT 960 with the \u03a6LSN DST assay provides a high-efficiency and low-cost method for TB DST simultaneously against multiple drugs within 3\u00a0days only, presenting potential to enhance the diagnosis and management of drug-resistant TB, especially in the resource-limited regions.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s40249-026-01470-5","type":"journal-article","created":{"date-parts":[[2026,6,16]],"date-time":"2026-06-16T05:23:56Z","timestamp":1781587436000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Rapid drug susceptibility testing of Mycobacterium tuberculosis against first-line drugs simultaneously all-in-one plate by using a novel high-sensitive reporter phage combined with the BACTEC MGIT 960 system"],"prefix":"10.1186","volume":"15","author":[{"given":"Ruiqing","family":"Ma","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ying","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Minghua","family":"Zhu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Muyang","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chengcheng","family":"Qian","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juan","family":"Wu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhidong","family":"Hu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mingquan","family":"Guo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiao-Yong","family":"Fan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,6,16]]},"reference":[{"key":"1470_CR1","unstructured":"World Health Organization. Global tuberculosis report 2025. 2025. http:\/\/www.who.int\/publications\/i\/item\/9789240101531. Accessed 21 Nov 2024."},{"issue":"10","key":"1470_CR2","doi-asserted-by":"publisher","first-page":"617","DOI":"10.1038\/s41579-024-01025-1","volume":"22","author":"M Farhat","year":"2024","unstructured":"Farhat M, Cox H, Ghanem M, Denkinger CM, Rodrigues C, Abd El Aziz MS, et al. Drug-resistant tuberculosis: a persistent global health concern. Nat Rev Microbiol. 2024;22(10):617\u201335.\u00a0","journal-title":"Nat Rev Microbiol"},{"issue":"5","key":"1470_CR3","doi-asserted-by":"publisher","first-page":"lxaf100","DOI":"10.1093\/jambio\/lxaf100","volume":"136","author":"X Mao","year":"2025","unstructured":"Mao X, Wang J, Xu J, Xu P, Hu H, Li L, et al. Current diagnosing strategies for Mycobacterium tuberculosis and its drug resistance: a review. J Appl Microbiol. 2025;136(5):lxaf100.","journal-title":"J Appl Microbiol"},{"key":"1470_CR4","unstructured":"World Health Organization. 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