Establishment And Application Of BRV And BCoV SYBR Green I RT-qPCR Detection Methods

Calf diarrhea is is one of the major diseases that cause yak’s growth and development and death.Pathogenic microorganisms accounts for about 75%-95% of the incidence of diarrhea in calves,among which Bovine rotavirus(BRV),Bovine coronavirus(BCoV)and Escherichia coli(E.coli)Mainly.In this study,BRV and BCoV SYBR Green I real-time quantitative PCR(RT-q PCR)detection methods were established,and the epidemiological investigations of the main pathogens(BVV,BCoV and E.coli)of diarrhea in calves in Heilongjiang were conducted.The PCR results of BRV and BCoV plasmids showed bands at 190 bp and 346 bp,respectively,which were consistent with the target gene fragments.The sequencing results showed homology with HQ09 VP6 gene and BCoV 2012/07 N gene by NCBI homology alignment results 100%.The established BRV and BCoV RT-q PCR methods have good specificity,stability and sensitivity.BRV RT-q PCR and RT-PCR methods were used to spot-check 46 feces specimens of diarrhea diarrhea in newborn calves.The positive rates were30.4%(14/46)and 19.6%(9/46),respectively.BCoV RT-q PCR and RT-PCR methods were used simultaneously.A total of 46 fresh yak diarrhea faecal samples were collected.The positive rates were 10.8%(5/46)and 19.5%(9/46),respectively.Epidemiological investigation showed that the positive rates of BRV,BCoV,and pathogenic E.coli were 27.5%(22/80),11.35%(9/80),and44%(35/80),respectively.The detection rates of E.coli containing K99 and F41 virulence factors were 40%(14/35)and 5.7%(2/35).Statistical analysis showed that the probability of occurrence of diarrhea was significantly higher in farms with more than 500 breeding scales(P<0.05)and was higher in cattle farms below the scale of 500 animals;the probability of occurrence of diarrhea in yak cattle adopting separate breeding methods was significant(P<0.05).It was lower than the mixed feeding method and was one of the risk factors for diarrhea in calves(OR value>1).The incidence of diarrhea was significantly higher in the cold season(P<0.01)than in the warm season.This experiment established a BRV and BCoV SYBR Green I RT-q PCR assay that can be used for the diagnosis and epidemiological investigation of BRV and BCoV.