Establishment And Preliminary Application Of Taqman Double Real-time Fluorescence Quantitative PCR Method For Porcine Pseudorabies Virus GB And GE Genes

Porcine pseudorabies(Pseudorabies,PR)is a viral disease caused by porcine pseudorabies virus(PRV).It can infect pigs of all ages.Among them,the mortality rate of piglets after infection is as high as 80%-100%,which is serious Land affects the production of pig farms.In recent years,the emergence of PRV variants in large-scale pig farms in my country has made the occurrence and prevalence of pseudorabies increasingly serious,causing huge economic losses to the pig industry.Real-time fluorescent quantitative PCR(Quantitative Real-time PCR,qPCR)is a common detection method at present.This method has the advantages of rapidness,strong specificity,high sensitivity,and good stability.It has now been applied to pathogen detection,genetic The direction of expression difference analysis,disease diagnosis,drug effect evaluation,etc.In this study,based on the variation of PRV epidemic in Henan Province,specific primers and probes were designed for the conserved regions of gE and gB genes of PRV epidemic strains since 2012.Based on the designed primers and probes,a Taqman dual real-time fluorescent quantitative PCR method for porcine pseudorabies virus gE and gB genes was established,and the established Taqman dual realtime fluorescent quantitative PCR diagnostic method was applied in clinical practice to verify the feasibility of the method.The main research is as follows:Construction of recombinant plasmids for PRV gE and gB genes:Use the primers that amplify the full length of the PRV gE and gB genes stored in our laboratory to perform PCR amplification on the PRV genome,and the obtained target fragments are recovered and purified by the gel and cloned into the pMD-18-T-Vector vector,and then transformed into DH5?.Plasmid extraction,double enzyme digestion identification of the obtained recombinant plasmid,and sequencing identification.The results of nucleic acid agarose gel electrophoresis showed that the obtained bands were consistent with expectations;the sequencing results showed that the genetic sequence of the recombinant plasmid was 100%similar to the selected conservative regions of the PRV gE and gB genes,indicating the successful construction of PRV gE and gB Recombinant plasmid of gB gene.Establishment of Taqman real-time fluorescent quantitative PCR method for PRV gE and gB genes:Through comparative analysis of the gE and gB gene sequences of the PRV epidemic strains in GenBank since 2012,two pairs of specific primers and corresponding probes were designed by selecting the respective conservative regions.Using the constructed PRV gE and gB gene recombinant plasmids as templates,the reaction conditions were optimized based on the reaction conditions recommended by Premix Ex Taq fluorescent quantitative reagents,and the PRV gE and gB gene Taqman real-time fluorescent quantitative PCR methods were established respectively.The linear relationship results show that the amplification efficiency of the two methods is between 90%-110%,and the linear correlation coefficient is above 0.99;the specificity test results show that both methods can only amplify the corresponding genome template.,While other pathogens are not amplified;the sensitivity test results show that the PRV gE gene Taqman real-time fluorescent quantitative PCR method for PRV gE gene contains the lowest detection quantity of 2.74 copies/uL,which is higher than the ordinary PCR of PRV gE gene established by our laboratory The sensitivity is about 100 times higher.The minimum detection amount of the PRV gB gene Taqman real-time fluorescent quantitative PCR method for the template containing the PRV gB gene is 2.71 copies/uL,which is also about 100 times higher than the general PCR sensitivity of the PRV gB gene established in our laboratory;The stability test results showed that the inter-assay and intra-assay coefficients of variation of the two methods were both lower than 2%,indicating that the Taqman real-time fluorescent quantitative PCR method for PRV gE and gB genes was successfully established.Establishment of the Taqman dual real-time fluorescent quantitative PCR method for PRV gE and gB genes:On the basis of the two established fluorescent quantitative PCR methods,by optimizing the reaction conditions of gE and gB genes,a single-tube simultaneous amplification of PRV gE and gB genes Taqman dual real-time fluorescent quantitative PCR method was established.The linear relationship results show that the amplification efficiency of this method is between 90%-110%,and the linear correlation coefficient is above 0.99;the specific test results show that this method can only amplify templates containing gE and gB gene bases,and Other pathogens are not amplified;the sensitivity test results show that the minimum detection amount of PRV gE gene is 27.10 copics/uL,and the minimum detection amount of PRV gB gene is 2.74 copies/uL.The stability test results showed that the inter-assay and intra-assay repetition coefficients of variation of the two methods were both lower than 2%,indicating that the PRV gE and gB gene Taqman dual real-time fluorescent quantitative PCR method was successfully established.Preliminary application of real-time fluorescent quantitative PCR method:adopt the PRV gE and gB gene Taqman dual real-time fluorescent quantitative PCR method established in this study to detect the viral load in the organs of mice after attacking PRV,and use the established method To explore the relationship between the copy number of pseudorabies virus gene and virus titer.The results showed that:after the mice were infected with PRV,PRV virus appeared in the internal organs to varying degrees.The virus content in the brain and lungs was the largest,and the virus content in the liver was the least;and the PRV gE and gB genes were detected simultaneously by fluorescence quantitative methods.Found that the results are consistent,and successfully verified that the established PRV gE and gB gene Taqman dual real-time fluorescent quantitative PCR method is feasible.Then the fluorescence quantitative PCR detection and titer determination with different titers of PRV confirmed that the virus copy number and the virus titer have a good linear relationship(R2=0.9998),the linear equation is:Y=1.083X-1.602,Y is The logarithm of virus copy number[lg(copies/?L)],X is the negative logarithm of virus titer[-lg(TCID50/0.1ml)].The Taqman dual real-time fluorescent quantitative PCR detection method for PRV gE and gB genes established in this study not only provides a method for rapid identification of PRV,but also enriches the detection methods of PRV.