Establishment Of MPCR Method For The Identification Of Wild-type And Vaccine Viruses Of CSF And PR And Research On Its Purification

Classical swine fever and Pseudorabies as two different kinds of major animal diseases still exist in extensive swine farms, which directly or indirectly affect the healthy development of pig industry in China. Recently, with the continuing breakthroughs and innovations in comprehensive prevention and control measures and advancement of the diagnosis technology of CSFV and PRV laid the foundation for the purification of these diseases. At the same time, the purification of these two kinds of disease having been included in the national long-term plan for animal disease prevention and control(2012-2020). In order to explore the purification scheme of CSF and PR,the use of PRV-g E/g B-ELISA antibody detection to distinguish wild-type and vaccine viruses,to establish a multiplex PCR method for distinguish differential wild-type and vaccine viruses, this article provides a reference as a large-scale pig farms swine fever and pseudorabies purification.The main research contents are as follows:1. The establishment of a single PCR detection method for identification of Wild-Type and Vaccine Viruses of CSFV and PRV3' NTR in Lapinized vaccine strain was found been with T-rich insertion sequence independently according to the genome sequence comparation and analyse of CSF wild viruses,vaccine virus and near origin virus published in Genabank. Based on this,two pairs of specific primers were designed on the upper and lower ends of the insertion sequence. According to the PRV vaccine, most of the missing g E gene sequence and the design of the primer for the gene, can identify PR wild virus infection.The anneal temperature of polymerese chain reaction(PCR)was optimized.The sensitivity and specificity results showed that the minimum detection amounts of nucleic acid by the single RT-PCR were 2.2 pg( One pair of primers) and 1.7pg(Another pair of primers in a single RT-PCR),respectively. And no amplified bands of PRV,PRRSV,JEV,BVDV,PCV2 DNA/RNA were detected by these methods. In PRV single PCR detection, positive bands were detected from the Guizhou isolated wild strains, while no bands were detected from the CSFV, PRRSV, JEV and PCV2 isolated strains. Then the methods were used to detect 146 suspicious clinical samples,and the results showed that the positive rate of the mixed infection by wild-type viruses infections of CSF and PRV in breeding sows, fattening pigs, nursery pigs and suckling piglets were 6.3%(3/48),7.4%(2/27),8.3%(3/36),8.6%(3/35), respectively.The results of this study indicated that the single PCR methods of CSF and PR have high specificity, sensitivity, and good repetitity. This study could provides important reference value for the purification of CSFV in large-scale swine farms.2. Establishment and Preliminary Application of the two-plex RT-PCR of CSFV method and triplex PCR of PRV methodBased on the established single PCR method of wild-type virus of CSFV,vaccine virus of CSFV, PR Guizhou-DY, Bartha-K61 and SA215, genomic sequences of eighteen CSFV strains and ten PRV strains including wild-type and vaccine virus pubulished in Gen Bank were compared and analyzed, and two pairs of specific primers were designed for the differentiation of wild-type and vaccine viruses of CSFV, three pairs of specific primers were designed for differentiation of wild virus infection and vaccine strains of PRV. USing the positive virus nucleic acid as the template, the PCR amplification,reaction system and reaction conditions were optimized. The two-plex RT-PCR method which could simultaneously detect the wild-type and vaccine viruses of CSFV was established,meanwhile the triplex PCR method which could simultaneously detect PRV wild virus and vaccine virus was also established. The preliminary application test showed the detection results of clinical samples were consistent with that of single PCR. The minimum detected amounts of nucleic acid were as follows:10.4 pg(wild-type virus of CSF), 29.8 pg(vaccine virus of CSF), 8.6pg(PR Guizhou-DY),26.3pg(Bartha-K61) and 9.4pg(SA215),respectively.3. Establishment and Application of Five-Plex PCR Method For The Differentiation of Wild-Type and Vaccine Viruses of Classical Swine Fever and Pseudorabies VirusIn this experiment, the two-plex RT-PCR of CSFV method and the triplex PCR of PRV method have been established, based on these five pairs of primers, a multi-PCR method for the differentiation of wild-type and vaccine viruses of CSFV and PRV wassuccessfully established. The results of the specificity and sensitivity tests of the five-plex PCR showed that the minimum detected amounts of nucleic acid were as follows:6.2 pg(wild-type virus of CSF), 21.3 pg(vaccine virus of CSF), 5.8pg(PR Guizhou-DY),20.8pg(Bartha-K61) and 8.6pg(SA215),respectively,and the amplification results on PRRSV, JEV and BVDV detection were all negative. The method was used to detect 134 suspicious clinical samples,and the results showed that the positive rate of the mixed infection by wild-type and vaccine viruses infections of CSF and PR in breeding sows, fattening pigs, nursery pigs and suckling piglets were 2.6%(1/38),7.7%(2/26), 8.3%(3/36) and 8.8%(3/34),the five-plex PCR method could detect DNA and RNA virus synchronously. It provides technical support for the rapid and efficient differentiation of wild-type and vaccine viruses of CSF and PR, and also provides a new method to detect pathogen for the purification of CSFV and PRV by the successful establishment of the five-plex PCR method.4. Study on Purification Scheme and Application of Classical Swine Fever and Pseudorabies in Extensive Swine FarmsIn this experiment, according to the swine industry status of our country, the control measures and purification scheme of CSFV and PRV in scale swine farms were made from the aspects of immunization,surveillance,prevention, etc. The control measures and purification scheme were applied in Guizhou four large-scale pig 1118 parts of the field at different stages(Breeding sows,Suckling piglets,Fattening pigs,Nursery pigs) blood samples for inspection CSFV, PRV(g B and g E) protein tonsil samples by ELISA for primary purification eliminated and the suspicious pigs infected with CSFV and PRV using m PCR method to establish the differential diagnosis, experimental results show that: In the application of the scheme for the first time on the purification of large-scale pig purification detection,through the elimination of positive pigs and strengthen the immune combination,it was found CSFV and PRV antibody levels were elevated, wild virus infection rates decline. The establishment of the purification scheme and preliminary application for the swine herds achieve positive pre-out pseudorabies purification provide reference for scale swine farms.