Establishment Of Dual RT-qPCR Detection For CSFV And BVDV,and Molecular Biological Studies Of CSFV

Classical swine fever(CSF)is a highly contagious lethal infectious disease caused by classical swine fever virus(CSFV).At this stage,the prevention and control of swine fever is usually carried out by swine fever,(C strain)vaccination prevention,the vaccine is recognized as the world’s most effective and safe attenuated vaccine,the prevention and control of swine fever played an important role.However,CSFV and Bovine viral diarrhea virus(BVDV)belong to the genus Flaviviridae,pestivirus,and there is antigen-related and cross-reactivity.Two viruses under the natural conditions of infection pigs,the clinical symptoms and pathological changes similar to increase the difficulty of clinical diagnosis.In order to understand the prevalence of CSFV in Shandong Province and to better monitor CSFV.In this study,a double fluorescent RT-PCR(RT-qPCR)method was established,and the C,E0,E1 and E2 genes of CSFV were sequenced and sequenced.In order to establish a method for simultaneous detection of classical swine fever virus(CSFV)and bovine viral diarrhea virus(BVDV),the double RT-qPCR was established by designing specific primers and TaqMan probes based on Genbank published gene sequences,the recombinant plasmids of CSFV Nproand BVDV 5’-UTR were constructed for positive control.The sensitivity of CSFV and BVDV were 100 copies /μl.The method has good specificity and stability.The RT-qPCR method was used to detect 206 pig samples preserved in our laboratory,59 CSFV positive and BVDV were negative,The CSFV test results were consistent with the single CSFV RT-qPCR kit used in our laboratory.In this study,the whole detection process of RT-qPCR was less than 3h,and the aerosol contamination was avoided by the direct treatment of closed tube reaction.The advantages of the double fluorescent quantitative RT-PCR established in this study is simple,rapid,high sensitivity and good biosafety,which can be used for the differential detection of CSFV and BVDV in swine infection and BVDV-1 and BVDV-2 can be detected,,so as to two kinds of viruscross-contamination detection to provide a convenient and fast method.To take in vitro culture technology,a total of 26 samples were found to be positive from Jinan,Dezhou,Linyi,Taian,Rizhao,Zibo and Jining,which were inoculated in PK15 cells.As a result,26 CSFV strains were isolated and were named as SDJN-1,SDJN-4,SDJN-4,SDJN-5,SDDZ-1,SDDZ-2,SDDZ-2,SDDZ-1,SDDZ-1,SDDZ-1,SDDZ-2,SDDZ-SDX-2,SDQH-1,SDQ-1,SDX-1,SDX-1,SDX-1,SDX-SDRZ-1,SDZ-1,SDTA-1,SDTA-2,SDSS-1,SDLS-1.The real time RT-PCR was used to certify that what we got all belonged to the epidemic field strains.The sequences of 26 CSFV C,E0,E1 and E2 genes were amplified,cloned and sequenced by molecular cloning technique.The sequencing results were sequenced and analyzed by DNAStar software,we found that it was highly homogeneous among the 26 separated strains.When compared the 26 strains with other classical strains such as Shimen stain,HCLV strain,HEBZ strain and so on,the results showed that the isolated strains and HEBZ strain shared so high homology in nucleotide and amino acid sequence.However,the homology between the isolated strains and other classical strain was 83.8%-96.2% for nucleotide sequence and 88.6%-97.6% for deduced amino acid sequence,The results indicated that the isolated strains perhaps have a close relationship to the CSFV strains in HEBZ province.A total of 34 isolates were divided into two branches,and the Shimen strain,the HCLV vaccine strain and the Brescia strain were divided into two branches.The results showed that the isolates were divided into two branches.And 26 isolates were closely related to Parderborn and HEBZ strains.The CSFV39 strain was 2.2,and the Alfort / Tueginen strain and the SP01 strain were composed of two groups was 2.3,the results show that the study of26 strains of CSFV belonging to 2.1 subgroups of Shandong is the dominant strain.