Point-of-care testing of rpoB in Mycobacterium tuberculosis using multiply-primed-RCA coupled with CRISPR/Cas12a

Abstract

Purpose: Due to the serious threat of tuberculosis to global health and limitations of existing diagnostic methods, this study combined the CRISPR/Cas12a system with Multiply-primed-RCA (MRCA) technology for Mycobacterium tuberculosis Point-of-care Testing (POCT). Method: We utilized T4 and Taq DNA ligases, compared the effects of specific primers and random 6NS primers on the method, and integrated MRCA and the CRISPR-Cas12a system in one tube. By optimizing conditions such as the concentration of DNA ligase, the concentration of padlock probes, and the number of cycles, we finally established T4-MRCA-Cas12a and Taq-MRCA-Cas12a methods for both stepwise and one-step. Results: The limits of detection of the one-step T4/Taq-MRCA-Cas12a were 104aM and 103aM. With no cross-reactivity with DNA from other bacterial strains. The accuracy and specificity were 88 % and 100 % for T4-MRCA-Cas12a, and 96 % and 100 % for Taq-MRCA-Cas12a, respectively. Conclusion: We developed a POCT method that can directly identify MTB through the naked eye.

Keywords

• Multiply-primed-RCA
• CRISPR/Cas12a
• Mycobacterium tuberculosis
• POCT
• dsDNA