Establishment And Application Of A One-step Multiplex Real Time RT-PCR(rRt-PCR) Assay For The Simultaneous Detection Of ASFV,PRRSV,CSFV And PRV

African swine fever virus(ASFV),Porcine reproductive and respiratory syndrome virus(PRRSV),Classical swine fever virus(CSFV)and Pseudorabies virus(PRV)were four general and serious hazard porcine pathogens in pig industry in China.Especially ASF epidemic trend rising continuously,PRRSV,CSFV and PRRSV gene diversity resulted the sensitivity reducing of some existed detection methods.Meanwhile,the application of vaccine strains of above pathogens also have different degrees of interference to the detection results.Above all,This study aimed to establish an one-step multiplex TaqMan probe real time RT-PCR assay for the rapid and simultaneous detection of ASFV,CSFV,PRRSV and PRV.And this methods initial applied in the clinical samples detection.Which provide an accurate tools for the pathogens detection and diseases epidemiological investigation,and also further understand the prevalence of these diseases in Sichuan and the molecular characteristics of the epidemic strains.It provides reference for the prevention,control and purification of four important porcine disease.1.Establishment of a one-step multiplex TaqMan probe real time RT-PCR assay for the simultaneous detection of ASFV,CSFV,PRRSV and PRV.This study targeted as B646L gene of ASFV,ORF 6 gene of PRRSV,3’ UTR gene of CSFV and gE gene of PRV to design four pairs specificity primers and probes and constructed nucleic acids standards.The concentration of Prime Script RT Enzyme Mix,primers and probes of four virus,and the annealing temperature were optimized.The specificity,sensitivity and stability were tested,and we also detecting 255 known samples by one-step multiplex TaqMan probe RT-PCR method and 4 commercial real time PCR kits to evaluated the agreement.Results showed that the combination of primers of ASFV,CSFV,PRRSV and PRV were 0.1 μmol/L,0.1μmol/L,0.06 μmol/L,0.14 μmol/L respectively,probes of 4 pathogens were 0.05μmol/L,respectively,probes of 4 pathogens were 0.05 μmol/L,1 μL(20U/μL)Prime Script RT Enzyme Mix Ⅱ,3μL nucleic acids,and annealing at 58℃;Specificity testing results indicated that only ASFV,CSFV,PRRSV and PRV could be detected correctly,but the C strains of CSFV,PRV(Bartha-k61)strain,BVDV and 10 other porcine pathogens could not be detected,which indicated that the one-step multiplex TaqMan probe RT-PCR methods has good specificity;Sensitivity testing results showed that the detection limits of this methods were:ASFV 3.41 × 101 copies/μL,CSFV 4.02 ×101 copies/μL,PRRSV 5.91 copies/μL,PRV 3.92 ×102copies/μL,respectively;Repeatability testing results showed that the coefficient of variation(CV)of CT value of this methods all less than 2.0%.It was indicated the good repeatability and stability of the one-step multiplex TaqMan probe RT-PCR within and between groups;the agreement rates between two methods were:ASFV 100%(k=1),CSFV 100%(k=1),PRRSV 100%(k=1),and PRV 100%(k=1).2.The epidemiological investigation of four important porcine disease in Sichuan province in 2020～2021 and genetic variation analysis of PRV wild strains.The one-step multiplex TaqMan probe real time RT-PCR assay was used to detect for PRRSV,CSFV and PRV in 293 clinical samples in 13 piggery in Bazhong,Dazhou,Nanchong,Guangan,Neijiang,Guangyuan and Yibin 7 areas.The clinical samples of piglets were 90,adult pigs were 203.Results showed that there were no detection of CSFV,and the positive rates of PRRSV and PRV were 7.8%(23/293)and 13.3%(39/293)respectively.The PRRSV only detected in Yibin and Guangyuan,and the positive rates of PRRSV in Yibin was achieved to 34.8%.But PRV were detected in 7 piggery in 5 areas except Yibin and Guangyuan.The positive rates of piggerys were achieved 53.84%(7/13).Results indicated that the PRRSV and PRV were widespread in Sichuan,and it’s necessary to strengthen prevention and control and purification measures in regions.The gB gene,gD gene and gE gene in positive samples in Schuan between 2020～2021 were amplified and sequenced by PCR methods.And the homology,amino acid variation,and genetic evolution were analysisd.Results showed that the gB,gC,gD and gE genes of 5 wild strains were sequenced in this study.The nucleotides homology of main virulence genes of the 5 wild PRV strains with all strains on NCBI were:gB gene 94.9～99.0%,gC gene 90.1～99.7%,gD gene 96.9～99.2%and gE gene 93.1～100%,respectively.The amino acid homology of main virulence proteins of the 5 wild PRV strains with all strains on NCBI were:gB protein 95.3～99.2%、gC protein 89.6～99.6%、gD protein 97.1～98.8%and gE protein 93.3～100%.There were multiple unique mutations of amino acid sites in gB protein of 5 isolated strains.The number were:3 of SWUN01,6 of SWUN02,8 of SWUN03,7 of SWUN04,5 of SWUN05,but only SWUN02 in 38aa and 39aa and SWUN05 in 137aa occur to amino acid mutate.And the protean analysis results also showed some amino acid mutations resulted in the change of antigenicity index.But there were only a few unique amino acids mutation in gD and gE protein.The Genetic evolutionary analysis of gB,gC and gE showed that the 5 wild strains were belong to group 1 and in a same branch with pandemic strains in China recent years but far away to isolated strains in SiChuan Province earlier.Moreover,the isolated strains in this study also in a same branch with Japanese and Korean strains but faraway to the strains in Europe and the United States,and it was speculated that the 5 strains may have similar evolutionary association.