A CRISPR-Cas12a-based platform for ultrasensitive rapid highly specific detection of Mycobacterium tuberculosis in clinical application

Abstract

Tuberculosis, caused by Mycobacterium tuberculosis (MTB), is the second leading cause of death after COVID-19 pandemic. Here, we coupled multiple cross displacement amplification (MCDA) technique with CRISPR-Cas12a-based biosensing system to design a novel detection platform for tuberculosis diagnosis, termed MTB-MCDA-CRISPR. MTB-MCDA-CRISPR pre-amplified the specific sdaA gene of MTB by MCDA, and the MCDA results were then decoded by CRISPR-Cas12a-based detection, resulting in simple visual fluorescent signal readouts. A set of standard MCDA primers, an engineered CP1 primer, a quenched fluorescent ssDNA reporter, and a gRNA were designed targeting the sdaA gene of MTB. The optimal temperature for MCDA pre-amplification is 67°C. The whole experiment process can be completed within one hour, including sputum rapid genomic DNA extraction (15 minutes), MCDA reaction (40 minutes), and CRISPR-Cas12a-gRNA biosensing process (5 minutes). The limit of detection (LoD) of the MTB-MCDA-CRISPR assay is 40 fg per reaction. The MTB-MCDA-CRISPR assay does not cross reaction with non-tuberculosis mycobacterium (NTM) strains and other species, validating its specificity. The clinical performance of MTB-MCDA-CRISPR assay was higher than that of the sputum smear microscopy test and comparable to that of Xpert method. In summary, the MTB-MCDA-CRISPR assay is a promising and effective tool for tuberculosis infection diagnosis, surveillance and prevention, especially for point-of-care (POC) test and field deployment in source-limited regions.

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

“…The specificity of TB One-Pot for diagnosing TB is 96.7%, which is consistent with the reported range of 94.1% to 100% in previous studies ( 4 – 8 , 21 – 24 ). In this study, among nine NTM samples, eight tested negative using the TB One-Pot method, while one showed a positive result, possibly indicating a co-infection of NTM and Mtb (Tables S1 and S2).…”

“…1f ). This LOD surpasses that of Huang et al’s CRISPR-TB method (0.25 copies/μL) ( 5 ) but remains below the typical range of LODs reported in various CRISPR-based TB detection studies (1–298 copies/μL) ( 4 – 8 , 21 – 24 ). Considering the variation in the number of IS 6110 copies in different Mtb complex strains’ genomes, related to strain evolution ( 31 ), we employed a single-copy plasmid template by cloning the IS 6110 target segment into the pUC57 plasmid.…”

“…3d ). While TB One-Pot’s LOD is lower than in the majority of CRISPR-based TB studies, its diagnostic sensitivity (67.2%) falls below the reported range of 78.4% to 100% in previous CRISPR-based TB studies ( 4 – 8 , 21 – 24 ). Several potential factors might explain this difference.…”

“…There have been nine published studies utilizing CRISPR Cas protein trans-cleavage activity for TB detection ( 4 – 8 , 21 – 24 ). These studies employed a two-step approach, involving the pre-amplification of the target sequence using techniques such as PCR, LAMP, or RPA, followed by the transfer of the amplified products to the CRISPR reaction system.…”

“…However, this approach raises concerns about the potential for cross-contamination and operational complexity. To our knowledge, current CRISPR-based TB detection systems all involve a two-step process ( 4 – 8 , 21 – 24 ). To address these limitations, we have developed an innovative TB detection method, which we have named TB One-Pot.…”