Abstract:
Objective To establish a multi-enzyme isothermal rapid amplification (MIRA) assay combined with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a for the detection of Yersinia pestis, and evaluate its sensitivity, specificity, and applicability in detection of clinical samples of plague patients and animal samples.
Methods Two target genes, pla and caf1, were selected for the development of the MIRA-CRISPR assay. Primers and probes of the pla gene were specially designed to ensure the specificity of Y. pestis detection by anchoring the 3' terminal nucleic acid to the Y. pestis-specific single nucleotide polymorphism (SNP) locus. The sensitivity of the assay was evalauted by using positive plasmid templates, while the specificity was evaluated by using non-Yersinia pestis DNA templates. The applicability of the assay was tested by detecting 6 blood or lymph samples of plague patients and 6 rodent liver samples from plague natural foci.
Results The optimized MIRA-CRISPR assay can finish the detection within 30 minutes. When the volume ratio of MIRA to CRISPR reached 1∶2, the detection sensitivity was optimal. The limit of detection of the MIRA-CRISPR assay was 9 copies for caf1 and 2 copies for pla. The spedificity test indicated that the MIRA-CRISPR assay can distinguish Y. pestis from other Yersinia. In the confirmation test of positive samples and negative contols, all the 6 positive human samples and 6 animal samples were detected, exhibiting excellent practicality.
Conclusion The MIRA-CRISPR assay developed in this study has high sensitivity and specificity, so it is suitable for the detection of human and animal plague, especially in samples with low target DNA content.
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