Establishment And Application Of Three Multiplex Fluorescent PCR Detection Methods For Important Diarrhea Pathogens In Swines,Poultry And Bovines

As important economic animals,swines,bovines and poultry meet the consumption demand of residents for various animal products.With the rapid development of domestic animal husbandry,large-scale farms have become the core subject of livestock and poultry production.While the scale level has been rapidly improved,animal diseases have also occurred frequently.Livestock and poultry diarrhea is a common disease in the breeding process with high incidence,which poses a great threat to the economic activities of animal husbandry and the production order of animal products.There are many kinds of diarrhea pathogens that can infect livestock and poultry,such as porcine epidemic diarrhea virus(PEDV),transmissible gastroenteritis virus(TGEV)and porcine deltacoronavirus(PDCo V),which cause porcine diarrhea.As well as Salmonella(SE),Escherichia coli(E.coli)and Clostridium perfringens(CP)causing avian diarrhea.Bovine astrovirus(BAst V),bovine viral diarrhea virus(BVDV)and infectious bovine rhinotracheitis virus(IBRV),which cause bovine diarrhea.These nine pathogens are harmful in the process of breeding and foreign trade,and the corresponding host disease performance is similar.Diarrhea is the main clinical symptom,and mixed infection occurs from time to time.Diarrhea is the main clinical symptom,and mixed infection occurs from time to time.Prevention and control has become a major problem in livestock and poultry breeding.Therefore,it is of great significance to establish a rapid,accurate,efficient and sensitive combined diagnosis method for the rapid detection of pathogens and the prevention and control of animal diarrhea.In this study,PEDV,TGEV and PDCo V triple fluorescent PCR methods,Salmonella,Escherichia coli and Clostridium perfringens triple fluorescent PCR methods,BAst V,BVDV and IBRV triple fluorescent PCR methods were established respectively.The amplification procedure and reaction conditions of the methods were optimized respectively.The constructed methods were applied to clinical samples inspection,and compared with the general PCR methods.At the same time,the pathogenic infection was analyzed to provide reference for the prevention and control of epidemic diseases.The results are as follows :1.The establishment and application of triple fluorescent PCR method for PEDV,TGEV and PDCo VIn order to establish a triple fluorescent PCR method for the differential detection of PEDV,TGEV and PDCo V,specific primers and probes were designed for the conserved sequences of PEDV ORF3 gene,TGEV N gene and PDCo V ORF1 ab gene,respectively.The PCR amplification procedure and reaction conditions were optimized,and the method was used to detect clinical samples.The results showed that the method had no cross-reaction with porcine circovirus(PCV),porcine parvovirus(PPV)and classical swine fever virus(CSFV),and had high specificity.The limits of detection(LOD)of PEDV,TGEV and PDCo V were 199 copies/μL.The coefficient of variation(CV)values of intra-and inter-assay repeatability tests were less than 3%,and the repeatability was good.Using this method to detect 25 clinical samples,the positive infection rates of PEDV,TGEV and PDCo V were 6.82%,0.76% and 0,respectively,and the test results were consistent with general PCR.2.Establishment and application of triple fluorescent PCR method for avian Salmonella,Escherichia coli and Clostridium perfringensIn order to establish a triple real-time PCR method for the identification and detection of Salmonella,Escherichia coli and Clostridium perfringens,the specific primers and probes were designed according to the sequence of Salmonella inv A,Escherichia coli pho A and Clostridium perfringens plc gene,and the PCR amplification procedure and reaction conditions were optimized.The method was used to detect clinical samples.The results showed that the method had no cross reaction with Pasteurella,Newcastle disease virus and Riemerella anatipestifer,had high specificity.The LODs of Salmonella,Escherichia coli and Clostridium perfringens was 66.9copies/μL,which was 100 times,1000 times and 1000 times higher than that of general PCR for Salmonella,Escherichia coli and Clostridium perfringens,respectively.The CV values intra-and inter-assay were lower than 3.85%,indicating good repeatability.By detecting 80 clinical samples and 23 identified strains,the results showed that the coincidence rate of the detection method with genaral PCR and sequencing results was 100 %..3.Establishment and application of a triple fluorescent PCR method for BAst V,BVDV and IBRVIn order to establish a rapid fluorescence detection method for BAst V,BVDV and IBRV,specific primers and probes were designed for the conserved regions of the ORF1 a gene of BAst V genome,the polyprotein gene of BVDV genome and the g B gene of IBRV genome.The amplification procedure and reaction conditions were optimized,and the specificity,sensitivity and repeatability tests were carried out.The method was used to detect clinical samples.The results showed that the method could specifically amplify BAst V,BVDV and IBRV nucleic acids,and had no cross-reaction with bovine coronavirus(BCV),lumpy skin disease virus(LSDV)and bovine Pasteurella(Pm).The LODs of BAst V,BVDV and IBRV were 305 copies/μL,and the sensitivity was high.The CV values of intra-and inter-assay repeatability tests of BAst V,BVDV and IBRV were less than 3%,and the repeatability was good.Using this method to detect 100 clinical samples,the positive infection rates of BAst V,BVDV and IBRV were 0,11% and 6%,respectively.The detection rates of BVDV and IBRV were significantly higher than those of general PCR.In summary,this study successfully constructed PEDV,TGEV and PDCo V triple fluorescence PCR methods,Salmonella,Escherichia coli and Clostridium perfringens triple fluorescence PCR methods,BAst V,BVDV and IBRV triple fluorescence PCR methods.These three methods have good specificity,sensitivity and stability,and can be effectively applied to the clinical sample diagnosis of the above-mentioned livestock and poultry diarrhea pathogens,providing technical support for epidemiological investigations.