Establishment and evaluation of a naked-eye diagnostic assay for tuberculosis utilizing reverse isothermal amplification-assisted CRISPR-Cas in resource-limited settings
Introduction: 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...
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2025-05-01
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| author | Ankush Kaushik Jitendra Singh Zeeshan Fatima Saif Hameed |
| author_facet | Ankush Kaushik Jitendra Singh Zeeshan Fatima Saif Hameed |
| author_sort | Ankush Kaushik |
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Introduction: 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 (spu
tum, 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 readout
based 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.
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| format | Article |
| id | doaj-art-a4e5c622680d416d8b89dc9595e45cce |
| institution | OA Journals |
| issn | 1177-3928 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | AboutScience Srl |
| record_format | Article |
| series | Drug Target Insights |
| spelling | doaj-art-a4e5c622680d416d8b89dc9595e45cce2025-08-20T02:28:32ZengAboutScience SrlDrug Target Insights1177-39282025-05-0119110.33393/dti.2025.3304Establishment and evaluation of a naked-eye diagnostic assay for tuberculosis utilizing reverse isothermal amplification-assisted CRISPR-Cas in resource-limited settingsAnkush Kaushik0Jitendra Singh1Zeeshan Fatima2Saif Hameed3Amity Institute of Biotechnology, Amity University Haryana, Gurugram (Manesar), Haryana - IndiaDepartment of Translational Medicine, All India Institute of Medical Sciences, Bhopal - IndiaAmity Institute of Biotechnology, Amity University Haryana, Gurugram (Manesar), Haryana - IndiaAmity Institute of Biotechnology, Amity University Haryana, Gurugram (Manesar), Haryana - India Introduction: 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 (spu tum, 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 readout based 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. https://journals.aboutscience.eu/index.php/dti/article/view/3304CRISPR-Cas12 Lateral flow readout Mycobacterium tuberculosis RT-LAMPTuberculosis |
| spellingShingle | Ankush Kaushik Jitendra Singh Zeeshan Fatima Saif Hameed Establishment and evaluation of a naked-eye diagnostic assay for tuberculosis utilizing reverse isothermal amplification-assisted CRISPR-Cas in resource-limited settings Drug Target Insights CRISPR-Cas12 Lateral flow readout Mycobacterium tuberculosis RT-LAMP Tuberculosis |
| title | Establishment and evaluation of a naked-eye diagnostic assay for tuberculosis utilizing reverse isothermal amplification-assisted CRISPR-Cas in resource-limited settings |
| title_full | Establishment and evaluation of a naked-eye diagnostic assay for tuberculosis utilizing reverse isothermal amplification-assisted CRISPR-Cas in resource-limited settings |
| title_fullStr | Establishment and evaluation of a naked-eye diagnostic assay for tuberculosis utilizing reverse isothermal amplification-assisted CRISPR-Cas in resource-limited settings |
| title_full_unstemmed | Establishment and evaluation of a naked-eye diagnostic assay for tuberculosis utilizing reverse isothermal amplification-assisted CRISPR-Cas in resource-limited settings |
| title_short | Establishment and evaluation of a naked-eye diagnostic assay for tuberculosis utilizing reverse isothermal amplification-assisted CRISPR-Cas in resource-limited settings |
| title_sort | establishment and evaluation of a naked eye diagnostic assay for tuberculosis utilizing reverse isothermal amplification assisted crispr cas in resource limited settings |
| topic | CRISPR-Cas12 Lateral flow readout Mycobacterium tuberculosis RT-LAMP Tuberculosis |
| url | https://journals.aboutscience.eu/index.php/dti/article/view/3304 |
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