{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,20]],"date-time":"2026-10-20T21:12:55Z","timestamp":1792530775294,"version":"4.5.0"},"reference-count":34,"publisher":"Aboutscience Srl","issue":"1","license":[{"start":{"date-parts":[[2025,5,9]],"date-time":"2025-05-09T00:00:00Z","timestamp":1746748800000},"content-version":"unspecified","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc\/4.0"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["dti"],"abstract":"<jats:p>\u00a0Introduction: The current scenario of tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) has presented an almost insurmountable challenge to hospitals with high patient numbers. Delayed diagnosis of TB is a major hurdle in preventing the employment of efficient therapeutics, leading to the development of drug resistance. Hence, an easily accessible diagnostic method, particularly for resource for resource-limited settings, is pertinent for the rapid identification of MTB-infected patients. In pursuit of developing such an assay, the present study offers a CLAP-TB (CRISPR-Cas coupled RT-LAMP Amplification Protocol for Tuberculosis) assay, which will allow us to diagnose TB rapidly and visually. Methods and results: Herein, the visual MTB detection consists of a method utilizing 232 different samples (sputum, urine, serum) from 82 patients for reverse transcription loop-mediated isothermal amplification (RT-LAMP). Additionally, the assay also utilizes the integration of a CRISPR-Cas12-based system using different guide RNAs of IS6110 and an internal control POP7 (human RNase P) genes along with visual detection via lateral flow readoutbased dipsticks with the unaided eye (~134 min). Overall, the limit of detection for CLAP-TB assay was up to 1 ag of RNA, while the clinical sensitivity and specificity were 98.27% and 100%, respectively, on the pilot scale.Conclusion: Together, our CLAP-TB assay offers proof of concept for a rapid, sensitive, and specific method with the minimum technical expertise required for TB diagnosis in developing and resource-limited settings.<\/jats:p>","DOI":"10.33393\/dti.2025.3304","type":"journal-article","created":{"date-parts":[[2025,5,9]],"date-time":"2025-05-09T07:04:57Z","timestamp":1746774297000},"page":"31-40","source":"Crossref","is-referenced-by-count":4,"title":["Establishment and evaluation of a naked-eye  diagnostic assay for tuberculosis utilizing reverse  isothermal amplification-assisted CRISPR-Cas in  resource-limited settings"],"prefix":"10.33393","volume":"19","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2819-4655","authenticated-orcid":false,"given":"Ankush","family":"Kaushik","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jitendra","family":"Singh","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zeeshan","family":"Fatima","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6477-1870","authenticated-orcid":false,"given":"Saif","family":"Hameed","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"18642","published-online":{"date-parts":[[2025,5,9]]},"reference":[{"key":"16450","unstructured":"World Health Organization. Global tubercolosis report 2024. Online https:\/\/www.who.int\/teams\/global-programme-on-tuberculosis-and-lung-health\/tb-reports\/global-tuberculosis-report-2024 (Accessed September 2024)"},{"key":"16451","doi-asserted-by":"crossref","unstructured":"Thakku SG, Lirette J, Murugesan K, et al. Genome-wide tiled detection of circulating Mycobacterium tuberculosis cell-free DNA using Cas13. Nat Commun. 2023;14(1):1803. https:\/\/doi.org\/10.1038\/s41467-023-37183-8 PMID:37002219","DOI":"10.1038\/s41467-023-37183-8"},{"key":"16452","doi-asserted-by":"crossref","unstructured":"Sandhu GK. Tuberculosis: current situation, challenges and overview of its control programs in India. J Glob Infect Dis. 2011;3(2):143-150. https:\/\/doi.org\/10.4103\/0974-777X.81691 PMID:21731301","DOI":"10.4103\/0974-777X.81691"},{"key":"16453","doi-asserted-by":"crossref","unstructured":"Thakur G, Thakur S, Thakur H. Status and challenges for tuberculosis control in India - Stakeholders\u2019 perspective. Indian J Tuberc. 2021;68(3):334-339. https:\/\/doi.org\/10.1016\/j.ijtb.2020.10.001 PMID:34099198","DOI":"10.1016\/j.ijtb.2020.10.001"},{"key":"16454","doi-asserted-by":"crossref","unstructured":"Lee JY. Diagnosis and treatment of extrapulmonary tuberculosis. Tuberc Respir Dis (Seoul). 2015;78(2):47-55. https:\/\/doi.org\/10.4046\/trd.2015.78.2.47 PMID:25861336","DOI":"10.4046\/trd.2015.78.2.47"},{"key":"16455","doi-asserted-by":"crossref","unstructured":"Purohit M, Mustafa T. Laboratory Diagnosis of extra-pulmonary tuberculosis (EPTB) in resource-constrained setting: state of the art, Challenges and the Need. J Clin Diagn Res. 2015;9(4):EE01-EE06. https:\/\/doi.org\/10.7860\/JCDR\/2015\/12422.5792 PMID:26023563","DOI":"10.7860\/JCDR\/2015\/12422.5792"},{"key":"16456","doi-asserted-by":"crossref","unstructured":"Wang Y, Wang Y, Ma AJ, et al. Rapid and sensitive isothermal detection of nucleic-acid sequence by multiple cross displacement amplification. Sci Rep. 2015;5(1):11902. https:\/\/doi.org\/10.1038\/srep11902 PMID:26154567","DOI":"10.1038\/srep11902"},{"key":"16457","doi-asserted-by":"crossref","unstructured":"Kohli M, Schiller I, Dendukuri N, et al. Xpert\u00ae MTB\/RIF assay for extrapulmonary tuberculosis and rifampicin resistance. Cochrane Database Syst Rev. 2018;27;8(8):CD012768. https:\/\/doi.org\/10.1002\/14651858.CD012768.pub2.","DOI":"10.1002\/14651858.CD012768.pub2"},{"key":"16458","doi-asserted-by":"crossref","unstructured":"Obande GA, Banga Singh KK. Current and future perspectives on isothermal nucleic acid amplification technologies for diagnosing infections. Infect Drug Resist. 2020;13:455-483. https:\/\/doi.org\/10.2147\/IDR.S217571 PMID:32104017","DOI":"10.2147\/IDR.S217571"},{"key":"16459","doi-asserted-by":"crossref","unstructured":"Wu D, Kang J, Li B, et al. Evaluation of the RT-LAMP and LAMP methods for detection of Mycobacterium tuberculosis. J Clin Lab Anal. 2018;32(4):e22326. https:\/\/doi.org\/10.1002\/jcla.22326 PMID:28940325","DOI":"10.1002\/jcla.22326"},{"key":"16460","doi-asserted-by":"crossref","unstructured":"Koonin EV, Makarova KS, Zhang F. Diversity, classification and evolution of CRISPR-Cas systems. Curr Opin Microbiol. 2017;37:67-78. https:\/\/doi.org\/10.1016\/j.mib.2017.05.008 PMID:28605718","DOI":"10.1016\/j.mib.2017.05.008"},{"key":"16461","doi-asserted-by":"crossref","unstructured":"Aman R, Mahas A, Mahfouz M. Nucleic acid detection using CRISPR\/Cas biosensing technologies. ACS Synth Biol. 2020;9(6):1226-1233. https:\/\/doi.org\/10.1021\/acssynbio.9b00507 PMID:32159950","DOI":"10.1021\/acssynbio.9b00507"},{"key":"16462","doi-asserted-by":"crossref","unstructured":"Sharma A, Agarwal A. Evaluation of the performance of a novel sputum processing ReaSLR methodology for culture of sputum samples in solid and liquid media in comparison with modified Petroff\u2019s method. Indian J Tuberc. 2021;68(2):255-260. https:\/\/doi.org\/10.1016\/j.ijtb.2020.09.015 PMID:33845961","DOI":"10.1016\/j.ijtb.2020.09.015"},{"key":"16463","doi-asserted-by":"crossref","unstructured":"Bhatt A, Fatima Z, Ruwali M, et al. CLEVER assay: a visual and rapid RNA extraction-free detection of SARS-CoV-2 based on CRISPR-Cas integrated RT-LAMP technology. J Appl Microbiol. 2022;133(2):410-421. https:\/\/doi.org\/10.1111\/jam.15571 PMID:35396760","DOI":"10.1111\/jam.15571"},{"key":"16464","doi-asserted-by":"crossref","unstructured":"Broughton JP, Deng X, Yu G, et al. CRISPR-Cas12-based detection of SARS-CoV-2. Nat Biotechnol. 2020;38(7):870-874. https:\/\/doi.org\/10.1038\/s41587-020-0513-4 PMID:32300245","DOI":"10.1038\/s41587-020-0513-4"},{"key":"16465","doi-asserted-by":"crossref","unstructured":"Tsou JH, Leng Q, Jiang F. A CRISPR test for detection of circulating nucleic acids. Transl Oncol. 2019;12(12):1566-1573. https:\/\/doi.org\/10.1016\/j.tranon.2019.08.011 PMID:31634698","DOI":"10.1016\/j.tranon.2019.08.011"},{"key":"16466","doi-asserted-by":"crossref","unstructured":"Ren W, Zhou Y, Li H, et al. Development and clinical evaluation of a CRISPR\/Cas13a-based diagnostic test to detect Mycobacterium tuberculosis in clinical specimens. Front Microbiol. 2023;14:1117085. https:\/\/doi.org\/10.3389\/fmicb.2023.1117085 PMID:36819015","DOI":"10.3389\/fmicb.2023.1117085"},{"key":"16467","doi-asserted-by":"crossref","unstructured":"Cao Y, Wu J, Pang B, et al. CRISPR\/Cas12a-mediated gold nanoparticle aggregation for colorimetric detection of SARS-CoV-2. Chem Commun (Camb). 2021;57(56):6871-6874. https:\/\/doi.org\/10.1039\/D1CC02546E PMID:34169944","DOI":"10.1039\/D1CC02546E"},{"key":"16468","doi-asserted-by":"crossref","unstructured":"Garcia-Venzor A, Rueda-Zarazua B, Marquez-Garcia E, et al. SARS-CoV-2 direct detection without RNA isolation with loop-mediated isothermal amplification (LAMP) and CRISPR-Cas12. Front Med (Lausanne). 2021;8:627679. https:\/\/doi.org\/10.3389\/fmed.2021.627679 PMID:33681254","DOI":"10.3389\/fmed.2021.627679"},{"key":"16469","doi-asserted-by":"crossref","unstructured":"Ai JW, Zhou X, Xu T, et al. CRISPR-based rapid and ultra-sensitive diagnostic test for Mycobacterium tuberculosis. Emerg Microbes Infect. 2019;8(1):1361-1369. https:\/\/doi.org\/10.1080\/22221751.2019.1664939 PMID:31522608","DOI":"10.1080\/22221751.2019.1664939"},{"key":"16470","doi-asserted-by":"crossref","unstructured":"Lu R, Wu X, Wan Z, et al. Development of a novel reverse transcription loop-mediated isothermal amplification method for rapid detection of SARS-CoV-2. Virol Sin. 2020;35(3):344-347. https:\/\/doi.org\/10.1007\/s12250-020-00218-1 PMID:32239445","DOI":"10.1007\/s12250-020-00218-1"},{"key":"16471","doi-asserted-by":"crossref","unstructured":"Yan L, Zhou J, Zheng Y, et al. Isothermal amplified detection of DNA and RNA. Mol Biosyst. 2014;10(5):970-1003. https:\/\/doi.org\/10.1039\/c3mb70304e PMID:24643211","DOI":"10.1039\/c3mb70304e"},{"key":"16472","doi-asserted-by":"crossref","unstructured":"Wang Y, Li J, Li S, et al. LAMP-CRISPR-Cas12-based diagnostic platform for detection of Mycobacterium tuberculosis complex using real-time fluorescence or lateral flow test. Mikrochim Acta. 2021;188(10):347. https:\/\/doi.org\/10.1007\/s00604-021-04985-w PMID:34542728","DOI":"10.1007\/s00604-021-04985-w"},{"key":"16473","doi-asserted-by":"crossref","unstructured":"Huang T, Zhang R, Li J. CRISPR-Cas-based techniques for pathogen detection: Retrospect, recent advances, and future perspectives. J Adv Res. 2023;50:69-82. https:\/\/doi.org\/10.1016\/j.jare.2022.10.011 PMID:36367481","DOI":"10.1016\/j.jare.2022.10.011"},{"key":"16474","doi-asserted-by":"crossref","unstructured":"Sam IK, Chen YY, Ma J, et al. TB-QUICK: CRISPR-Cas12b-assisted rapid and sensitive detection of Mycobacterium tuberculosis. J Infect. 2021;83(1):54-60. https:\/\/doi.org\/10.1016\/j.jinf.2021.04.032 PMID:33951419","DOI":"10.1016\/j.jinf.2021.04.032"},{"key":"16475","doi-asserted-by":"crossref","unstructured":"Yang X, Huang J, Chen Y, et al. Development of CRISPR\/Cas12b-based multiple cross displacement amplification technique for the detection of Mycobacterium tuberculosis complex in clinical settings. Microbiol Spectr. 2023;11(2):e0347522; Epub ahead of print. https:\/\/doi.org\/10.1128\/spectrum.03475-22 PMID:36975805","DOI":"10.1128\/spectrum.03475-22"},{"key":"16476","doi-asserted-by":"crossref","unstructured":"Mao X, Xu M, Luo S, et al. Advancements in the synergy of isothermal amplification and CRISPR-cas technologies for pathogen detection. Front Bioeng Biotechnol. 2023;11:1273988. https:\/\/doi.org\/10.3389\/fbioe.2023.1273988 PMID:37885449","DOI":"10.3389\/fbioe.2023.1273988"},{"key":"16477","doi-asserted-by":"crossref","unstructured":"Yigci D, At\u00e7eken N, Yetisen AK, Tasoglu S. Loop-mediated isothermal amplification-integrated CRISPR methods for infectious disease diagnosis at point of care. ACS Omega. 2023;8(46):43357-43373. https:\/\/doi.org\/10.1021\/acsomega.3c04422 PMID:38027359","DOI":"10.1021\/acsomega.3c04422"},{"key":"16478","unstructured":"Khan MA, Mirza SH, Abbasi SA, et al. Peripheral blood-based polymerase chain reaction in diagnosis of pulmonary tuberculosis. J Ayub Med Coll Abbottabad. 2006;18(2):25-28. PMID:16977809"},{"key":"16479","doi-asserted-by":"crossref","unstructured":"Thabet S, Souissi N. Transposition mechanism, molecular characterization and evolution of IS6110, the specific evolutionary marker of Mycobacterium tuberculosis complex. Mol Biol Rep. 2017;44(1):25-34. https:\/\/doi.org\/10.1007\/s11033-016-4084-x PMID:27766569","DOI":"10.1007\/s11033-016-4084-x"},{"key":"16480","doi-asserted-by":"crossref","unstructured":"Gao H, Shang Z, Chan SY, et al. Recent advances in the use of the CRISPR-Cas system for the detection of infectious pathogens. J Zhejiang Univ Sci B. 2022;23(11):881-898. https:\/\/doi.org\/10.1631\/jzus.B2200068 PMID:36379609","DOI":"10.1631\/jzus.B2200068"},{"key":"16481","doi-asserted-by":"crossref","unstructured":"Bumbrah GS, Jain S, Fatima Z, et al. Efficacy of LAMP assay for Mycobacterial spp. detection to prevent treatment delays and onset of drug resistance: a systematic review and meta-analysis. Drug Target Insights. 2023;17:78-89. https:\/\/doi.org\/10.33393\/dti.2023.2596 PMID:37304408","DOI":"10.33393\/dti.2023.2596"},{"key":"16482","doi-asserted-by":"crossref","unstructured":"Thakku SG, Lirette J, Murugesan K, et al. Genome-wide tiled detection of circulating Mycobacterium tuberculosis cell-free DNA using Cas13. Nat Commun. 2023;14(1):1803. https:\/\/doi.org\/10.1038\/s41467-023-37183-8 PMID:37002219","DOI":"10.1038\/s41467-023-37183-8"},{"key":"16483","doi-asserted-by":"crossref","unstructured":"Patel K, Nagel M, Wesolowski M, et al. Evaluation of a urine-based rapid molecular diagnostic test with potential to be used at point-of-care for pulmonary tuberculosis: Cape Town cohort. J Mol Diagn. 2018;20(2):215-224. https:\/\/doi.org\/10.1016\/j.jmoldx.2017.11.005 PMID:29269279","DOI":"10.1016\/j.jmoldx.2017.11.005"}],"container-title":["Drug Target Insights"],"original-title":[],"link":[{"URL":"https:\/\/journals.aboutscience.eu\/index.php\/dti\/article\/download\/3304\/4014","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/journals.aboutscience.eu\/index.php\/dti\/article\/download\/3304\/4015","content-type":"application\/epub+zip","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/journals.aboutscience.eu\/index.php\/dti\/article\/download\/3304\/4014","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,5,9]],"date-time":"2025-05-09T07:05:12Z","timestamp":1746774312000},"score":1,"resource":{"primary":{"URL":"https:\/\/journals.aboutscience.eu\/index.php\/dti\/article\/view\/3304"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,5,9]]},"references-count":34,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2025,1,13]]}},"URL":"https:\/\/doi.org\/10.33393\/dti.2025.3304","relation":{},"ISSN":["1177-3928"],"issn-type":[{"value":"1177-3928","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,5,9]]}}}