Establishment And Application Of Multiplex PCR For The Diagnosis Of Major Viral Diseases In The Digestive Tract Of Porcine

In recent years,Porcine digestive tract viroid disease is the most common and prominent disease in pig breeding practice,which has brought great harm and loss to pig industry in China,which in china has brought great harm and loss to pig breeding industry.Many studies at home and abroad have shown that: porcine epidemic diarrhea virus(PEDV),porcine transmissible gastro enteritis virus(TGEV),porcine deltacoronavirus(PDCoV),porcine rotavirus a(PoRVA),and porcine rotavirus C(PoRV C)are the main viral pathogens that can cause diarrhea in the digestive tract of pigs.It often presents mixed infection or secondary infection,resulting in high rate of morbidity and mortality in pigs,and making the diagnosis and prevention much more difficult.In order to bring out a rapid detection method for the diagnosis of the mixed infection of various pathogens in the clinical porcine digestive tract viral diseases,this study established a five plex RT-PCR for the above pathogens and put the method into preliminary application,providing an effective technical means and theoretical reference for the rapid diagnosis and control of the pathogens of the viral diseases in the digestive tract of pigs.The main research contents are as follows: 1.The establishment and detection of single RT-PCR methods for PEDV?TGEV?PDCoV?PoRVA and PoRV CRefer to partial fragments of genes,PEDVN(KT323979),TGEVN(EU074218),PDCoVN(MH715491),PoRVAVP6(MH320796)and PoRV CVP6(KJ814472)etc.on Gen Bank,by using related software and platforms such as primerselect,five pairs of primers were designed to amplify partial fragments of PEDVN,TGEVN,PDCoVN,PoRVAVP6 and PoRV CVP6 genes,the length of them were 799 bp,375 bp,271 bp,525 bp,184 bp respectively.The fecal samples and intestinal tissues which have been preserved and collected in the laboratory and tested positive for PEDV,TGEV and PoRVA,and the partial fragments of PDCoVN gene and PoRV CVP6 gene were synthesized by the biological company to obtain the positive clone bacterium liquid and plasmid.The c DNA template of PEDV,TGEV and PoRVA was transcriptied from the RNA which was extracted from the positive samples,meanwhile the positive clones of PDCoVN gene and PoRV CVP6 gene were extracted as templates.By identification of the products amplified by single RT-PCR of five viruses,it was found that the amplification results were consistent with the expectations.The RT-PCR products of PEDV,TGEV and PoRVA were recovered and linked with p MD-19 T vector,then transformed into the receptive cells.After screening the positive clones,the corresponding recombinant plasmids were extracted.Five kinds of recombinant plasmids were used as reaction templates to optimize the annealing temperature of single PCR reaction,and the sensitivity was tested.The single RT-PCR methods for PEDV,TGEV,PDCoV,PoRVA and PoRV C detection were established.The establishment of single RT-PCR detection methods for each of the viruses showed that:(1)the optimal reaction system: 5 pairs of primers were 10 ?mol/L,upstream and downstream primers of TGEV,PDCoV and PoRV C each 1 ?L,while PEDV and PoRVA 1.5 ?L each,the template was 2 ?L each,and with the gold Mi X(Green)up to 25 ?L.(2)Optimum reaction conditions: the specific annealing bands of single PCR for each virus can be amplified at 51.0?60.5°C,but 55.1? was the best choice.The optimal reaction procedures for each of the viruses are: 94? 5 min(Pre denaturation);94? 45 s(denaturation);55.1? 45 s(annealing);72? 45 s(extension),35 cycles;72?10 min(extension)(3)Sensitivity results showed that the minimum nucleic acid detection amounts of the viruses were: PEDV 0.39 pg,TGEV 2 0.34 pg,PDCoV 0.34 pg,PoRVA 0.37 pg,PoRV C 0.34 pg.2.The establishment and detection of five-plex RT-PCR methods for PEDV?TGEV?PDCoV?PoRVA and PoRV CBased on the established single RT-PCR of PEDV,TGEV,PDCoV,PoRVA and PoRV C,by selecting the appropriate five-plex RT-PCR reaction system,including the optimization of annealing temperature,the dosage of templates and primers.Lastly,the specificity,sensitivity and repeatability of the five-plex RT-PCR were detected to establish a rapid and simultaneous identification of PEDV,TGEV,PDCoV,PoRVA and PoRV C five-plex RT-PCR detection methods for five common swine digestive tract viroid diseases.The results showed that:(1)the volume of 50 ? l was the best reaction system of RT-PCR.The two-step PCR system was as follows: the working concentration of the upstream and downstream primer was all 10 ?mol/L,and the dosages of primers were TGEV,PDCoV,PoRV C each 0.75?L,PEDV and PoRVA each 1 ?L,the dosages of the templates: 1 ?L for each of TGEV,PDCoV and PoRV C,for PEDV and PoRVA each 1.5 ?L,and up to 50 ?L with Gold Mi X(Green);the one-step system was as follows: the working concentration of the upstream and downstream primer was all 10 ?mol/L,and the dosages of primers were TGEV,PDCoV,PoRV C each 0.75?L,PEDV and PoRVA each 1 ?L,the dosages of the templates: 1 ?L for each of TGEV,PDCoV and PoRV C,for PEDV and PoRVA each 1.5 ?L,the rest of the system was made up with 2×1 Step Buffer 25 ?L,Prime Script One Step Enzyme Mix 2 ?L and RNase free water 8.5 ?L.(2)Optimum reaction conditions: 94? for 5 min;94? 5 min(Pre denaturation);94? 45 s(denaturation);55.1? 45 s(annealing);72? 45 s(extension),35 cycles;72?10 min(extension).(3)S The results of specificity,sensitivity and repeatability tests showed that: five-plex RT-PCR could amplify specific fragments of PEDV,TGEV,PDCoV,PoRVA and PoRV C,which are 799 bp,375 bp,271 bp,525 bp and 184 bp respectively,while the DNA/RNA of CSFV,JEV,PCV2,PRV and normal cell tissues were not amplified;the minimum detection amount of nucleic acid for each virus in the five-plex PCR was PEDV 39 pg,TGEV 34 pg,PDCoV 35 pg,PoRVA 37 pg and PoRV C 34 pg.3.Application of PEDV?TGEV?PDCoV?PoRVA and PoRV C Five-plex RT-PCR Detection Methods in Clinical SamplesThe five-plex RT-PCR method for the main common digestive tract viruses in pigs was used to detect 523 clinical samples collected from different areas,large-scale pig farms and farmers in Guizhou Province during 2017-2020.The results showed that the positive rate of virus detection were PEDV 26.96%(141/523),PoRVA respectively 13.96%(73/523),TGEV 1.72%(9/523),PDCoV 1.53%(8/523)and PoRV C had not been detected.the double infection rate of PEDV+TGEV was 1.72%(9/523),the double infection rate of PEDV+PDCoV was 1.53%(8/523),the double infection rate of PEDV+ PoRVA was 1.91%(10/523).No triple infection or more was detected.The results of five-plex RT-PCR were consistent with those of single RT-PCR.