Development of a CRISPR-Cas12a based assay for the detection of swine enteric coronaviruses in pig herds in China

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 × 10⁴ Relative Fluorescence Units (RFU), 1.57 × 10⁴ RFU, 3.07 × 10⁴ RFU and 1.64 × 10⁴ 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.

Keywords

Swine enteric coronaviruses / CRISPR-Cas12a / Nucleocapsid gene / Detection assay

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Funding

Key-Area Research and Development Program of Guangdong Province(2022B1111030001); National Key Research and Development Program, China(2021YFD1801101)