AI Article Synopsis
- This study combines CRISPR/Cas12a technology with Multiply-primed-RCA (MRCA) for improved point-of-care testing (POCT) for tuberculosis, addressing limitations in current diagnostic methods.
- Researchers optimized the method using different DNA ligases and primers, ultimately creating two effective testing methods (T4-MRCA-Cas12a and Taq-MRCA-Cas12a) that can be performed in a single tube.
- The testing methods showed high accuracy and specificity, effectively detecting tuberculosis with no interference from other bacterial DNA, allowing for results to be observed visually.
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 Point-of-care Testing (POCT).
Method: We utilized T4 and Taq DNA ligases, compared the effects of specific primers and random 6N 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 10aM and 10aM. 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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Source |
|---|---|
| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC11416492 | PMC |
| http://dx.doi.org/10.1016/j.heliyon.2024.e37640 | DOI Listing |
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