Abstract:
Objective To establish a rapid and sensitive detection method for Coxsackievirus A10 (CVA10) by combining Multienzyme Isothermal Rapid Amplification (MIRA) with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and its associated protein Cas13a, and evaluate its performance by using clinical samples from hand foot and mouth disease (HFMD) cases.
Methods Specific MIRA amplification primers and CRISPR RNAs (crRNAs) were designed and screened targeting the conserved VP1 region of CVA10. Two detection modalities, a fluorescence assay and a lateral flow strip assay, were established by using specific fluorescent probes. A one-pot reaction system was established and optimized by using overlaying mineral oil to prevent contamination. The sensitivity and specificity of both methods were systematically evaluated. Furthermore, the reproducibility of the fluorescence assay and its clinical consistency with real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) were evaluated.
Results The minimum detection limit for both real-time fluorescence and test strip methods was 0.50 copies/μL. The methods demonstrated good specificity with no cross-reactivity to common enteric and non-enteric viruses. The precision was high, with an intra-assay variation coefficient of <10.00%. Clinical sample validation demonstrated good consistency with RT-qPCR (Kappa=0.94), with a positive consistency rate of 100.00% and a negative consistency rate of 94.00%.
Conclusion The MIRA-CRISPR/Cas13a CVA10 nucleic acid detection assay established in this study has good consistency with RT-qPCR. It is an effective technical approach for rapid field screening and primary-level laboratory detection of CVA10-associated HFMD.
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