Rapid Identification of High-Prevalence Drug-Resistant Mutations in <i>Mycobacterium tuberculosis</i> Using a Portable Colorimetric Array

The emergence and rise of drug-resistant mutations in Mycobacterium tuberculosis (TB) exacerbates the challenges of TB controlling and treatment. 95% of the TB-related deaths occurred in low- and middle-income countries (LMICs), where there is a lack of access to quality diagnostics for drug-resistant mutations. Here, we present an approach for rapid identification of 24 high-prevalence drug-resistant mutations in M. tuberculosis, in which a portable thermal cycler and a photosensitization colorimetric assay (PCA) were employed for polymerase chain reaction (PCR) and subsequent analysis of the PCR amplicons, respectively. To avoid potential “off-target” effects for mutation detection, we introduced two reverse primers (binding two regions) to ensure the successful amplification of PCR. An analysis of 50 clinical samples revealed that our method achieved a sensitivity of 100%, a specificity of 99.48%, and an accuracy of 99.50%, with the results of the clinical approach serving as the standard reference. Furthermore, a portable colorimetric array (PoCA) was designed to streamline the operations of PCA, thereby increasing the number of detectable mutations per round and enhancing the feasibility of drug-resistant mutation detection in LMICs. To facilitate real-world applications, uracil-DNA glycosylase (UDG) and deoxyuridine triphosphate (dUTP) were integrated into the PCR system, thereby reducing potential cross-contamination. Due to its easy accessibility, high accuracy, and rapid turnaround time, this approach offers a promising option for large-scale identification of drug-resistant mutations in M. tuberculosis.