Study On Technology Of Detecting Pathogenic Nucleic Acids In Porcine Viral Diarrhea Diseases

Porcine viral diarrhea is a contagious intestinal infectious disease.Its clinical symptoms are vomiting,diarrhea and dehydration.The pathogens include Porcine Bocavirus(PBCo V),Norovirus(Norovirus,No V),Porcine Delta Coronavirus(Porcine Delta Coronavirus,PDCo V),porcine variant diarrhea virus(Porcine Bocavirus,PBCo V),rotavirus(swine)and porcine transmissible gastroenteritis virus.Among the six viruses,the clinical symptoms,epidemiological laws and pathological changes caused by these viruses are very similar.In particular,porcine diarrhea virus,its variant strains continue to appear,the prevention and control technology is more demanding,and it is difficult to distinguish its pathogens in clinical diagnosis.Therefore,establishing a same reaction,rapid and sensitive detection method will be beneficial to the disease.Prevention and control.It is not easy to distinguish pathogens from clinical diagnosis.Therefore,establishing a same reaction,rapid and sensitive detection method will be conducive to the prevention and control of the disease.In this study,Multiplex Ligation-dependent Probe Amplification(MLPA)and fluorescence quantitative RT-PCR technology were used to establish MLPA detection methods for differentiating 6 viral diarrhea pathogens and fluorescent quantitative RT-PCR method for porcine variant diarrhea strains and vaccine strains,so as to provide technical support for rapid detection of pathogens of porcine viral diarrhea diseases.1.Study on MPLA technology for detection of six pathogens of porcine viral diarrheaMLPA is a new high throughput technique for qualitative and quantitative analysis of specific target sequences in various pathogenic nucleic acids.In this study,using MLPA technology,PBCo V,No V,PDCo V,PEDV,RV and TGEV were taken as the research objects.MLPA probes for six viruses were designed respectively.According to the procedures of denaturation,annealing,hybridization,amplification and detection,MLPA methods for identical reaction and simultaneous detection of six viruses were established.The results showed that when the concentration of primer probe was 1.33 nm/L,the probe of each virus had only a single amplification peak for its own template,and its size was basically consistent with the prediction.The probe had good amplification efficiency and had no specific amplification peak for other viral templates;only a single amplification peak was amplified for each diarrhea pathogen in the six probes mixed system,in the same system.At the same time,the specific peaks of 6 pathogens were obtained,but no common viruses were found in pig farms such as Porcine Reproductiveand Respiratory Syndrome Virus(PRRSV),swine fever virus(Classical Swine Fever Virus,CSFV),pseudorabies virus(Fever),and porcine circovirus type 2(2).Sex amplification peak.The minimum detection limits of MLPA for six pig diseases were RV(75 ng/response),PEDV(7.53 ng/response),TGEV(7.49 ng/response),PDCo V(7.54 ng/response),No V(7.56 ng/response),PBCo V(75.8 ng/response).According to the established MLPA method,52 clinical samples were tested,and negative and positive controls were set up.The results showed that the positive rate of PEDV was 47.06%(95% CI: 19.9%-26.9%),RV was 1.96%(95% CI: 0-10.4%),TGEV was 1.96%(95% CI: 0-10.4%)and the mixed infection rate of PEDV,RV and TGEV was 1.96%(95% CI: 0-10.4%).No other viruses were detected.The six viral MLPA detection methods established in this study have the purpose of one sampling,one analysis and simultaneous detection of six porcine viral diarrhea diseases.This method has the advantages of strong specificity,high sensitivity and multiple detection,and is expected to become a new technology for animal disease detection in the future.2.Establishment of one-step double fluorescence quantitative RT-PCR for detection of PEDV variant strains and vaccine strainsA pair of primers and two specific probes were designed according to the S gene sequence of 8 typical variant strains of swine variant diarrhea virus(PEDV)and 5 strains of vaccine strain(G I)released by Gen Bank.The mutated strain carried FAM fluorophore,and the vaccine strain carried HEX fluorophore group.A one-step double fluorescent quantitative RT-PCR detection method based on Taq Man probe was established.The results showed that within the range of 10-1-108 copies per_L,the method had a good amplification efficiency and a good linear relationship between the Ct value and the concentration of G II strain(R2=0.9892)and G I strain(R2=0.9914);the detection sensitivity was 7.388 copies per strain of G II strain,4.322 copies per strain of G I strain;there was no cross-reaction between G I strain and G II strain,and there was no cross-reaction between the method and C.There was no cross-reaction among CSFV,TGEV,RV,PRV,JEV,PPV and other pathogens,and the specificity was good.The coefficient of variation between groups and within groups was low and the repeatability was good.The kappa value of 12 clinical samples was 0.75 by one-step RT-PCR compared with that of common industry standard method,and 12 samples were identified as positive by virus isolation.The results showed that 12 samples were PEDV.The mutant strain was positive,and the coincidence rate with PEDV virus isolation was 100%.The one-step dual fluorescence quantitative RT-PCR method based on Taq Man probe has the advantages of sensitivity,specificity,high throughput and accurate quantification,and can be used for rapid differential diagnosis of PEDV variant strains and vaccine strains.The MLPA method for detection of various porcine viral diarrhea viruses was established,and 6 pathogens of porcine viral diarrhea virus were detected.The pathogen is fast,sensitive,specific and reproducible.It provides high throughput detection technology for the surveillance and emergency diagnosis of porcine viral diarrhea virus.Compared with conventional fluorescence quantitative RT-PCR method,the one-step double real-time fluorescence quantitative PCR method based on Taqman probe in this study makes the detection results more accurate and more sensitive,and is conducive to identifying the prevalence of PEDV mutant strains and vaccine strains in clinical samples.The method established in this study laid a technical foundation for the clinical diagnosis,prevention and control of viral diarrhea in pig farms.