Rapid detection of Mycobacterium tuberculosis in sputum using CRISPR-Cas12b combined with cross-priming amplification in a single reaction

ABSTRACT The diagnosis of tuberculosis (TB) still lacks a rapid, user-friendly method. Recent research has revealed the significant diagnostic potential of the CRISPR system in TB detection. However, most current CRISPR-based diagnostic tests involve a two-step process, including nucleic acid amplification followed by CRISPR-guided sequence-specific detection, which raises concerns about cross-contamination and operational complexity. In this study, we introduced TB One-Pot, which combines CRISPR-Cas12b-mediated trans-cleavage with cross-priming amplification, enabling one-pot detection in a single reaction. TB One-Pot achieves a minimum detection limit of 0.8 copies/μL for H37Rv genomic DNA and a quantification limit of 50 CFU/mL. It exhibited no cross-reactivity with common nontuberculous mycobacteria and respiratory pathogens. TB One-Pot employs real-time fluorescence, enabling the process from nucleic acid extraction to result in reporting in just 80 minutes. It also includes a UV-based visual check, eliminating the need for specialized equipment. A retrospective cohort study evaluated the diagnostic performance of this method using a composite reference standard as the gold standard. TB One-Pot demonstrated a sensitivity of 67.2% and an area under the curve (AUC) of 0.820 in sputum samples, surpassing conventional methods such as culture and acid-fast bacilli (AFB) smear. Comparative analysis with Xpert (sensitivity:64.2%, AUC:0.821) showed no statistically significant difference ( P > 0.05). TB One-Pot exhibited a specificity of 96.7%, similar to Xpert, culture, and AFB smear ( P > 0.05). TB One-Pot excels in speed, sensitivity, specificity, and practical requirements. Ongoing refinements aim to improve sensitivity, making it a promising solution for overcoming limitations in molecular TB diagnosis, particularly in resource-constrained settings. IMPORTANCE In this study, we successfully established a new One-Pot method, named TB One-Pot, for detecting Mtb in sputum by combining CRISPR-cas12b-mediated trans-cleavage with cross-priming amplification (CPA). Our study evaluated the diagnostic performance of TB One-Pot in clinical sputum samples for tuberculosis. The findings provide evidence for the potential of TB One-Pot as a diagnostic tool for tuberculosis.