Establishment Of The Triplex RT-PCR For The Detection Of PEDV,PdCV And TGEV

Porcine epidemic diarrhea virus(PEDV)and Transmissible gastroenteritis virus of swine(TGEV)werekinds of highly contagious intestinal infectious diseases,which resulted in diarrhea,dehydration,vomiting and loss of appetite.The pigs of all ages were susceptible,especially in the piglets.Furthermore,they had the seasonal variations.Porcine deltacorona virus(PdCV)was a novel porcine deltacoronavirus,which was first identified in Hong Kong in 2009.In addition,it was reported in Ohio and Indiana in February 2014,rapidly spread to other states in the United States and Canada.It had been confirmed that the PdCV could infect and cause the host of acute diarrhea.Recently,there were many cases of PdCV that had been detected in China,and the disease was widely spread and had a catastrophic economic impact on the pigs industry.PEDV,PdCV and TGEV often had similar clinical symptoms.All of them could cause piglets dehydration,vomiting and diarrhea,and were often co-infection.PdCV was related to the diarrhea epidemic at the same time.Therefore,we established a triplex RT-PCR method tosimultaneously detect the three viruses.According to the conserved genes M,N and S of PEDV,PdCV and TGEV,we designed and compounded three pairs of specific primers,then successfully amplified these three genes,and established the differential diagnosis of the three kinds of viruses for the first time.After optimized the amount of primers of PEDV,PdCV and TGEV,they were 10 pmol/L,10 pmol/L and 20 pmol/L respectively.When the annealing temperature was 53�C,the amplification effect was the best.The sensitivity assay showed that the triplex RT-PCR could detect 60.96 pg/reaction(8.16�103copies/reaction)of PEDV cDNA,58.85 pg/reaction(1.14�104copies/reaction)of PdCV cDNA and 102.69 pg/reaction(4.32�104copies/reaction)of TGEV cDNA.For the specificity test,all of pathogens in the negative controls were no amplicons.Finally,for the repeatability test,the results of positive detection ratewere consistent for 1 d,15 d,30 d and 60 d.Therefore,the results showed that the established triplex RT-PCR method had a good sensitivity,specificity and repeatability,and could be used for clinical testing.The triplex RT-PCR method and single RT-PCR method were used in clinical practice.During 2015~2016,196 samples were detected(176 feces samples of diarrhea pigs,20 fecal samples of healthy pigs)in Sichuan and Chongqing.In 176 feces samples of diarrhea pigs,the positive rate of PEDV,PdCV and TGEV were 57.4%,2.8% and 2.3% respectively.3 of these feces samples were co-infection of PEDV and PdCV,and the positive rate was 1.7%.In samples of 20 healthy pigs,the positive rate of PEDV and PdCV were 5.0%,respectively,but no positive sample of TGEV was detected.There was no co-infection.The coincidence rates were 95.3% of PEDV,100% of PdCV and 100% of TGEV,respectively.The above results verified that triplex RT-PCR method we established was accurate and specitic,and can be better used in clinical detection.Therefore,it provides an effective technical support for pathogenic molecular epidemiology investigation,Besides,we can further explore the relationship between thetraditional diarrhea pathogens and potential new intestinal pathogens.