Establishment And Preliminary Application Of Digital PCR Method For PDCoV And PEDV Detection

Porcine deltacoronavirus(PDCoV)and porcine epidemic diarrhea virus(PEDV)are both porcine enteropathogenic coronaviruses,which can cause acute diarrhea,vomiting,dehydration and even death in newborn piglets.Chip digital PCR(cd-PCR)is the third-generation PCR technology for water-in-oil droplets with special chips as carriers.It has more sensitive and accurate characteristics and can be applied to the early diagnosis of virus infection.Therefore,this experiment aims to establish an efficient and fast detection method using cd-PCR.It can not only detect PDCoV and PEDV accurately and quantitatively,but also monitor the virus aerosol formed by PDCoV or PEDV in real time,which provides technical support for virus purification and biosafety prevention and control in pig farms.The specific research contents of this experiment are as follows:1.Development and preliminary clinical application of single cd-PCR for detection of PDCoV and PEDV In this study,specific primers and probes for PDCoV and PEDV cd-PCR detection methods were designed according to the N gene of PDCoV and the M genetic sequence of PEDV published in Gen Bank,and the positive vectors pMD18-T-PDCoV-N and pMD18-T-PEDV-M.The test results show that when the annealing temperature of the PDCoV and PEDV singlet cd-PCR detection method is 58 ℃,and the concentration of primers and probes is 800 nM and1000 nM,the amplification effect is the best,the distance between positive droplets and negative droplets is the largest,and the positive droplets are the most concentrated,and the number of intermediate miscellaneous droplets is the smallest.After the constructed standard plasmid was diluted ten times the ratio layer,the detectable copies of the established method were 3.36 copies/μL and 2.12 copies/μL,respectively,with high sensitivity;the amplification results of common porcine viruses such as porcine transmissible gastroenteritis virus(TGEV),porcine sapelovirus(PSV)and porcine pseudorabies virus(PRV)were negative,indicating that the method had good specificity;the coefficient of variation of intra-group repeats and inter-group repeats were less than 7%,and the repeatability was good.The established PDCoV cd-PCR method was used to detect tissue samples(heart,liver,spleen,lung,kidney,submental lymph nodes,mesenteric lymph nodes,brain,cecum and jejunum)of PDCoV-infected piglets.The results showed that PDCoV can be widely distributed in various organs of the collected piglets after infection,which is consistent with the qPCR results.The PEDV cd-PCR detection method established in this experiment was used to detect 20 porcine diarrhea materials,and parallel tests were carried out with qPCR and RT-PCR.The results showed that the positive detection rates of PEDV by the three methods were 65%(13),50%(10)and 35%(7),respectively,indicating that the detection method has high sensitivity and can be applied to clinical detection.2.The establishment and preliminary clinical application of the dual cd-PCR detection method for PDCoV and PEDV In this study,specific primers and probes for cd-PCR were designed according to the N gene of PDCoV and the M gene of PEDV in Gen Bank.The test results show that when the annealing temperature is58 ℃ and the mixed primer probe system is 5 μL,the spacing between the positive droplets and the negative droplets of the two channels is the largest,and the positive droplets are the most concentrated,and the amplification effect is the best;the amplification results of common porcine viruses such as TGEV,PSV,and PRV are all negative,with good specificity;after the constructed standard plasmid is diluted ten times over the layer,single copies of the diluted plasmid are verified to be detectable,with good sensitivity;the significance of the analysis of variance results is greater than 0.05,and the accuracy is strong.The established dual cd-PCR method was used to detect 48 clinical porcine diarrhea samples,and the parallel test was carried out with the qPCR detection method.The results showed that the positive detection rates of PDCoV were 22.92%(11)and 12.5%(6),the positive detection rates of PEDV were 37.5%(18)and14.58%(7),and the detection rates of mixed infection were 10.42%(5)and 4.17%(2),respectively.3.Construction and preliminary clinical application of PDCoV and PEDV aerosol research models In order to construct a PDCoV and PEDV aerosol research model,a 1 m~3 enclosed space was first built with plastic film.PDCoV(4.25× 10~6copies/mL)and PEDV(7.6 × 10~6copies/mL)virus venom were mixed 1 mL each and diluted 10,100 and 1000 times,respectively.The aerosol generator was used to emit the virus mixture(for 6 minutes),collect virus particles in the air and enrich them.The PDCoV and PEDV dual cd-PCR detection method was used to calculate the virus copy number in the enrichment solution and analyze it.The results show that the constructed aerosol research model can successfully collect PDCoV(2352copies/m~3,5103 copies/m~3,15309 copies/m~3)and PEDV(1008 copies/m~3,19341copies/m~3,99603 copies/m~3)virions.In order to explore the production and distribution of viral aerosols around PDCoV-infected piglets,three 5-day-old healthy piglets were selected for PDCoV infection test(oral challenge titer was 1 × 10~8TCID50/,10 mL/head).The results showed that the content of PDCoV virions was the highest at 0 m(5218 copies/μL at24 h and 5870 copies/μL at 72 h),decreased at 2 m(338 copies/μL at 24 h and 1239copies/μL at 72 h),and decreased at 4 m(7 copies/μL at 24 h and 9 copies/μL at 72h).This experiment successfully established single and double cd-PCR detection methods for PDCoV and PEDV,providing a fast,accurate and efficient new method for clinical detection of PDCoV and PEDV.At the same time,the PDCoV and PEDV aerosol research model was successfully constructed,and the established cd-PCR detection method was used to analyze it.It was found that PDCoV and PEDV can form viral aerosols in the air,which provides a new research model for in-depth exploration of their transmission mechanism.