Ambient-Temperature Extraction-Free One-Pot CRISPR Detection of Mycobacterium tuberculosis

Background Current molecular diagnostics for Mycobacterium tuberculosis (MTB) require complex nucleic acid extraction procedures and laboratory infrastructure, limiting their use as point-of-care (POC) tests in resource-limited settings. To address these barriers, we developed an extraction-free one-pot CRISPR assay for rapid detection of MTB directly from minimally processed sputum specimens. Methods The assay integrates ambient-temperature chemical lysis, recombinase polymerase amplification, and CRISPR-Cas12a detection within a single closed-tube workflow. A conserved region of the MTB-specific IS6110 insertion sequence was targeted for detection. Analytical performance was evaluated using serially diluted MTB genomic DNA standards, followed by clinical validation using 100 archived sputum specimens, including 50 MTB-positive samples and 50 MTB-negative controls. Results The extraction-free assay detected MTB genomic DNA within 30 minutes and achieved an analytical limit of detection of 100 copies per reaction. In clinical validation, the assay correctly identified 48 of 50 MTB-positive specimens and 49 of 50 MTB-negative specimens, yielding 96.0% sensitivity and 98.0% specificity. Conclusions This study demonstrates the feasibility of extraction-free one-pot CRISPR-Cas12a detection of MTB directly from sputum specimens. By eliminating conventional nucleic acid extraction while maintaining high analytical sensitivity and diagnostic performance, the platform may facilitate future development of rapid molecular diagnostics for POC and resource-limited settings.