Nested PCR Assay for Detection of Bovine Coronavirus S1 Gene

Abstract

A nested PCR targeted to the S1 ectodomain gene of the spike (S) glycoprotein of coronaviruses was developed to detect bovine coronavirus (BCoV) in stool specimens from cattle. One outer and one internal pair of primers were designed to conserved regions surrounding the hypervariable region of the S gene (GenBank accession No. M31053). Outer primers produced an 885bp-long product (nn 1204 to 2088 from S gene) and the internal primers a 488bp-long product (nn 1329 to 1816 from S gene, internal to the first PCR). Optimal annealing temperatures were tested in a gradient thermocycler and used in the final protocols. Outer primers and the final nested protocol detection limits were tested with the BCoV Kakegawa strain diluted two-fold in BCoV-free stool suspension. The nested PCR assay was tested in 22 clinical stool specimens from calves with or without diarrhea and 1 sample from a diarrheic cow, resulting in 10 positive samples both from diarrheic and non-diarrheic individuals. The nested PCR reported here is a specific and sensitive tool for bovine coronavirus diagnosis.