猪德尔塔冠状病毒RT-PCR检测方法的建立及应用OA北大核心CSCDCSTPCD

This experiment was conducted to develop a sensitive and rapid detection method for porcine deltacoronavirus(PDCoV).A reverse transcription polymerase chain reaction(RT-PCR) for detecting PDCoV was established using a pair of specific primers based on the M gene (359 bp fragment) of PDCoV published in GenBank.Through the optimization of reaction components in the RT-PCR system,a sensitive and specific PDCoV RT-PCR assay was established,and its sensitivity,specificity and repeatability were tested.The results showed only PDCoV was successfully amplified with a target fragment of 359 bp in size,but the amplification results were negative for porcine reproductive and respiratory syndrome virus,classical swine fever virus,porcine epidemic diarrhea virus,transmissible gastroenteritis virus of swine,rabies virus,bovine viral diarrhea virus,porcine parvovirus,porcine circovirus type 2,pseudorabies virus,Streptococcus suis type 2 and Escherichia coli.Testing of the sensitivity of RT-PCR indicated that the lowest detection concentration was 1 ng/μL.The method was used to detect 110 clinically suspected cases of swine diarrhea,and the results showed the positive rate of PDCoV was 11.8％.Blast analysis showed homology between positive samples and other PDCoV nucleotide sequence was 92.2％ to 100％.Phylogenetic analysis based on M genes indicated that PDCoV strains had closer relationship with Chinese PDCoV strains than the strains isolated from other countries.The above result suggested that we successfully established an RT-PCR method for detection of PDCoV.The method can be used to monitor and epidemiologically investigate PDCoV infection.