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Updated: Jun 7, 2026
Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray
Published on: April 25, 2014
Point-of-care testing of rpoB in Mycobacterium tuberculosis using multiply-primed-RCA coupled with CRISPR/Cas12a
Yingying Sun1,2, Yaozhou Wu3, Yulin Wang4
1School of Public Health, Gansu University of Chinese Medicine, Lanzhou, 730000, PR China.
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.
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