Work

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

Year: 2024

Type: article

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 6N S 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 10 4 aM and 10 3 aM. 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.

Source: Heliyon

DOI: 10.1016/j.heliyon.2024.e37640

Authors: Yingying Sun, Yaozhou Wu, Yulin Wang, Keke Li, Yanbin Chang, Lianhua Wei

Institutions: Gansu University of Traditional Chinese Medicine, Gansu Provincial Hospital, Lanzhou University, Ningxia Medical University

Language: English

Topic: Biosensors and Analytical Detection

Subfield: Biomedical Engineering

Field: Engineering

Domain: Physical Sciences

SDG: Good health and well-being

Open Access status: gold

Estimated APC paid: $2,100