Establishment and evaluation of a naked-eye diagnostic assay for tuberculosis utilizing reverse isothermal amplification-assisted CRISPR-Cas in resource-limited settings

<div xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="section"> <a class="named-anchor" id="st1"> <!-- named anchor --> </a> <h5 class="section-title" id="d4671853e161">Introduction:</h5> <p dir="auto" id="d4671853e163">The current scenario of tuberculosis (TB) caused by <i>Mycobacterium tuberculosis</i> (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. </p> </div><div xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="section"> <a class="named-anchor" id="st2"> <!-- named anchor --> </a> <h5 class="section-title" id="d4671853e169">Methods and results:</h5> <p dir="auto" id="d4671853e171">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 <i>IS6110</i> and an internal control <i>POP7</i> (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. </p> </div><div xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="section"> <a class="named-anchor" id="st3"> <!-- named anchor --> </a> <h5 class="section-title" id="d4671853e180">Conclusion:</h5> <p dir="auto" id="d4671853e182">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. </p> </div>