Establishment And Preliminary Clinical Application Of Multiplex RT-PCR Assay For The Detection Of PDCoV,TGEV And PEDV

Porcine transmissible gastroenteritis virus(TGEV)and Porcine epidemic diarrhea virus(PEDV)are 2 major pathogens of viral diarrhea in swine,both of them belong to the alpha coronavirus of the genus coronavirus,the main symptoms are vomiting,dehydration,watery diarrhea,various ages of pigs are susceptible to infection,especially for suckling of the piglets have serious harm,the infection rate was almost up to 100%.Since 2010,the PED break out with a new feature in many provinces of China,the incidence of serious,high death rates,prevention and control are difficult,which suggest that the epidemic strains may have genetic variation and cause a great distress to the swine industry.PDCoV is a newly discovered diarrhea virus,belongs to the Delta coronavirus.The clinical symptoms of the PDCoV are similar to the TGE and PED,and the two virus are prone to mixed infection and difficult to distinguish in clinical application.At present,the virus has been detected in the United States and Canada,South Korea and other countries,and gradually expanding the scope of infection;attracting great attention to the global porcine industry.Therefore,it is of great significance to establish an effective,convenient and rapid diagnosis method for the prevention and control of viral diarrhea.Because the clinical features and pathological changes of PDCoV,TGEV and PEDV are very similar,but also mixed infections.It is difficult to distinguish these virus,so we need the help of laboratory diagnosis methods.The RT-PCR method has good sensitivity and specificity,and it is widely used in the detection of pathogens.Especially the multiplex RT-PCR method has the advantages of RT-PCR method,and can achieve the purpose of rapid and simultaneous identification of multiple pathogens.Therefore,this study established the multiplex RT-PCR differential diagnosis methods of the 3 viruses,which has good specificity and sensitivity.The specific research contents are as follows:1 This study according to the sequence of TGEV N gene,PEDV M gene and PDCoV N gene that published on the GenBank,then analysis the homology and the conserved sequence.we designed and synthesized 3 pairs of specific primers.By conventional RT-PCR reaction amplification reaction,the plastic recycling of PCR products,established a single RT-PCR diagnostic method to detect PDCoV,TGEV and PEDV respectively.And sensitivity and specificity were tested to establish the single RT-PCR method.The results showed that the minimum detection amount of TGEV is 368 copies/μL,PEDV is 388 copies/μL,and PDCoV is 314copies/μL;the PRRSV,PCV2 and PRV are negative.The results showed that the method established in this study can detect the 3 viruses effectively and accurately,which are beneficial to the early diagnosis and prevention of these 3 diseases.2 On the foundation of the establishment of which the single RT-PCR of PDCoV,PEDV,TGEV,through the optimization of PCR reaction system and reaction system,eventually successfully established the PDCoV+TGEV,PDCoV+PEDV and TGEV+PEDV duplex RT-PCR method.that using PDCoV+TGEV duplex reaction system can while spread increased out PDCoV of 383bp specific fragments and TGEV of 229bp specific fragments,the minimum detection amount respectively 314 copies/μL,3.68×103 copies/μL;PDCoV+PEDV duplex RT-PCR method,that using with RT-PCR reaction system can while spread increased out PDCoV of 383bp specific fragments and PEDV of 101bp specific fragments,the minimum detection amount respectively 3.14×103 copies/μL,3.88×104 copies/μL;TGEV+PEDV duplex RT-PCR method,that using with RT-PCR reaction system can while spread increased out TGEV of 229 bp specific fragments and PEDV of 10lbp specific fragments,the minimum detection amount respectively 368 copies/μL,388 copies/μL;The three methods was not detected in the target band syndrome of PRRSV,PCV2,PRV and negative control.It shows that the method is suitable for the detection and differential diagnosis of mixed infection of PDCoV,TGEV and PEDV.3 Based on the duplex RT-PCR method,we first established a PDCoV,TGEV and PEDV multiplex RT-PCR diagnostic method through the optimization of PCR reaction conditions and procedure in the domestic.This method can discern the specificity fragment of PDCoV 383bp,TGEV 229bp and PEDV 10lbp,The methods detected PRRSV,PCV2,PRV and negative control are negative..The minimum detectable amount of PDCoV,TGEV and PEDV was 3.14×103copies/μL,3.68×104 copies/μL and 3.88×104 copies/μL respectively.It is proved that this method is suitable for the detection and diagnosis of PDCoV,TGEV and PEDV mixed infection.