2.4 Nucleic Acid Detection of CoVs using a conserved RT-PCR assay
A conserved RT-PCR and sequencing was used to analysis the genetic characteristics of CoVs circulating in poultry. In brief, the RNA stored above were amplified with the Takara One Step RT-PCR Kit (Takara), using a conserved RT-PCR assay designed by our lab with the primers 5’-GGTTGGGATTAYCCWAARTGYGA-3’ (upper) and 5’-YTGTGAACAAAAYTCRTGWGGACC-3’(down). The assay amplifies a 550 bp-nucleotide region in the viral 1ab gene, and this assay has been proved to detect main CoV circulating in animals, including pigs, chickens, ducks, geese, pigeons, etc. The RT-PCR detection was performed in a 25-µl reaction system with incubation at 50 ℃ for 30 min and denaturation at 94 ℃ for 5 min, followed by 30 cycles at 94 ℃ for 30 s, 50 ℃ for 30 s and 72 ℃ for 45 s. RT-PCR products were purified with an agarose gel DNA extraction kit (Sangon, Shanghai, China), and sequenced directly using the ABI 3730xl DNA Analyzer for the following phylogenetic analysis.