A TaqMan-probe-based Multiplex Real-time RT-qPCR For Simultaneous Detection Of Porcine Enteric Coronaviruses

At present,viral diarrhea severely affects swine industry,causing tremendous economic loss worldwide.The younger the infected piglets are,the higher the mortality rate there will be.The main factors responsible for swine diarrhea include viruses,bacteria,poisoning,management,and etc.Among which,diarrhea caused by viral infection is common but more difficult to deal with.Enteric coronaviruses are the population of viral diarrhea.The traditional coronaviruses causing diarrhea include porcine epidemic diarrhea virus(PEDV),transmissible gastroenteritis virus(TGEV).In 2012,an emerged swine enteric virus,porcine deltacoronavirus(PDCoV)was discovered and circulating in pig farms.In late 2017,another novel porcine enteric Alphacoronavirus(PEAV)emerged in pig farms in Guangdong province.It is critically important but hard to make a differential diagnosis on porcine viral diarrhea due to complicated pathogens in the gastrointestinal tract and high similarity in clinical signs and pathological changes presented when diarrhea occurs.This study aimed to establish a specific and sensitive detection method for simultaneous detection of TGEV,PEDV,PDCoV,and PEAV in one reaction vial using real-time RT-qPCR technology.To accomplish this goal,specific primers and probes were designed based on the highly conserved regions of TGEV N,PEDV M,PDCoV M,and PEAV N genes,respectively.The singular real-time RT-qPCR for detection of each pathogen was first established.The results showed that the limit of detection for these four viruses can reach as low as 10 copies in singular real-time RT-qPCR assays.The multiplex real-time RT-qPCR was next established based on singular real-time RT-qPCR and optimized.The result showed that the multiplex real-time RT-qPCR assay can detect as low as 100 copies of the target gene,with correlation coefficients(R2)of all assays maintained at above 0.99,and the amplification efficiency was between 90%and 110%.The multiplex real-time RT-qPCR reaction was capable of specific detection of the four selected pig viruses,without cross-reaction with other non-targeted porcine viruses.The multiplex real-time RT-qPCR assay was then validated by detecting 407 field diarrheal samples collected from 21 pig farms located in 9 provinces of China during the time frame between October 2015 and July 2018.The highest positive rates and provinces of PEDV,PDCoV,TGEV,and PEAV were 40.2%in Chongqing,100%in Shandong,63.6%in Ningxia,and 80%in Shandong respectively.The co-infection of porcine enteric coronaviruses commonly existed in some pig farms,but the gene copy of TGEV and PEAV was very low.This well-established multiplex real-time RT-qPCR assay provided a rapid,efficient,specific and sensitive differential diagnostic tool for detection of four swine enteric coronaviruses,facilitating the diagnosis,identification,and treatment of swine viral diarrhea.The real-time RT-qPCR assay developed in this study is of great significance for the prevention and control of epidemic diseases and epidemiological investigation of swine viral diarrhea.