Abstract
Porcine epidemic diarrhea virus (PEDV), Transmissible gastroenteritis virus (TGEV), Porcine deltacoronavirus (PDCoV) and Swine acute diarrhea syndrome coronavirus (SADS-CoV) rank among the most frequently encountered swine enteric coronaviruses (SECoVs), leading to substantial economic losses to the swine industry. The availability of a rapid and highly sensitive detection method proves beneficial for the monitoring and surveillance of SECoVs. Based on the N genes of four distinct SECoVs, a novel detection method was developed in this study by combining recombinant enzyme polymerase isothermal amplification (RPA) with clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas) 12a. Results showed that the cut-off value of CRISPR-Cas12a assay for SADS-CoV, PEDV, PDCoV and TGEV was 2.19 × 10(4) Relative Fluorescence Units (RFU), 1.57 × 10(4) RFU, 3.07 × 10(4) RFU and 1.64 × 10(4) RFU, respectively. The coefficient of variation (CV) of within and between runs by CRISPR-Cas12a assay for 6 clinical diarrhea samples were both less than 10%. The CRISPR-Cas12a assay demonstrated high specificity for TGEV, PEDV, PDCoV, and SADS-CoV with no cross-reactivity to other common swine viruses. This method also exhibited a low limit of detection of 2 copies for each virus. Additionally, the results demonstrated a perfect agreement (100%) between the CRISPR-Cas12a assay and the RT-qPCR assay. Finally, a total of 494 pig samples from the field tested by CRISPR-Cas12a assay showed that positive rate for SADS-CoV, TGEV, PDCoV and PEDV was 0, 0, 1.2% and 48.6%, respectively. The results suggested the great potential of CRISPR-Cas12a assay to detect SECoVs in the field.