LAUSR

BACKGROUND Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread over the world since its emergence. Although the dominant route of SARS-CoV-2 infection is respiratory, a number of studies revealed infectious risk from contaminated surfaces and products, including porcine derived food and other products. The SARS-CoV-2 outbreak has been severely threatening public health, disrupting porcine products trade and pig industry. Swine acute diarrhea syndrome coronavirus (SADS-CoV), which was responsible for a large-scale of fatal disease in piglets emerging in 2017, has also caused enormous economic losses in pig industry. Currently, RT-rPCR is the gold standard method for SARS-CoV-2 diagnosis and most commonly used for SADS-CoV detection. However, the inaccurate detection for the SARS-CoV-2 infection obtained by RT-rPCR are increasingly reported, especially in specimens with low viral load. OBJECTIVE This study aimed to develop an accurate RT-ddPCR assay for the detection of SARS-CoV-2 and SADS-CoV simultaneously. METHODS Two pairs of primers and one double-quenched probe targeting the RDRP region of ORF1ab gene of SARS-CoV-2 and the corresponding ORF1ab region of SADS-CoV were designed to develop the RT-ddPCR assay. The sensitivity, specificity, repeatability, and reproducibility were tested by cRNAs and clinical specimens. RESULTS The detection limits of RT-ddPCR were 1.48 ± 0.18 and 1.38 ± 0.17 copies in a 20 μL reaction for SARS-CoV-2 and SADS-CoV cRNAs respectively (n = 8), showing approximately 4 and 10 fold greater sensitivity than RT-rPCR assay. This assay also exhibited good specificity, repeatability and reproducibility. CONCLUSION The established RT-ddPCR assay showed it to be a highly effective, accurate and reliable method for the sensitive detection of SARS-CoV-2 and SADS-CoV. HIGHLIGHTS This RT-ddPCR assay could be used to detect both SARS-CoV-2 and SADS-CoV in a sample with one double-quenched probe, and is also the first report of the establishment of an RT-ddPCR assay for SADS-CoV detection.