Establishment Of GeXP Multiplex PCR Assay For Detecting Six Cattle And Sheep Viral Pathogens

Goat pox virus(GTPV),Sheep pox virus(SPPV), Lumpy skin diseases virus (LSDV), Contagious ecthyma virus(CPDV), bluetongue virus(BTV), peste des petits ruminants virus(PPRV) can cause high incidence of acute febrile infectious diseases in ruminants. Except for CPDV, the remaining five viruses were OIE repoting epidemic viruses. Goat pox, sheep pox, lumpy skin diseases, bluetongue and peste des petits ruminants are classified as Class I animal disease and contagious ecthyma is Class III animal disease by the ministry of agriculture of China. In addition, goat pox, sheep pox, contagious ecthyma are zoonotic diseases. They can cause not only serious harm to animal husbandry and losses in economy, but also pose a serious hazard to human health. Currently, there are no reports of lumpy skin diseases epidemic in China. The clinical symptoms of diseases caused by these six viruses are similar, which cause herpes or ulcer on the shin or mucosa and they often has mixed infections. Therefore, it is difficult to distinguish the six kinds of diseases just only from clinical symptoms. Traditional diagnostic methods such as virus isolation and identification, serological methods are complicated operation, time consuming, low sensitivity and high false positive rate, which are difficult to detect multiple pathogens simultaneously. Therefore, for the clinic and rapid detection of ports, it is an urgent need to establish a rapid, accurate, sensitive and good stability detection method that can be used for detecting simultaneously the six pathogens as mentioned above.Gene eXpress Profiler-polymerase chain reaction (GeXP-PCR) is a new technology. The universal primers are mixed up with specific chimeric primers according to proper proportion for PCR and then the fragment length of amplification products treated by dye marker is measured by GeXP system.GeXP-PCR has the advantages of strong specificity, high sensitivity, high throughput. In recent years, the system is commonly used in gene expression analysis, virus detection and other areas of research and application.In this study, a method called GeXP multiplex PCR is established to detect GTPV, SPPV, LSDV, CPDV, BTV and PPRV. This method provides appropriate technical support for the diagnosis of six kinds of disease, disease surveillance, epidemiology, entry-exit animal quarantine. It is very important for protection the safety and healthy development of animal husbandry of China.The main contents and results of this thesis are as follows:(1)Six pairs of specific primers were designed according to the conserved sequence of GTPV, SPPV, LSDV, CPDV, BTV, PPRV. After PCR amplification, cloning and sequencing of the fragment, it was found that the homology of amplification products with the various strains in GenBank was over 97%, indicating that the six pairs of specific primers could amplify the target sequence.(2)Through the optimization of primer concentration and annealing temperature for the specific chimeric primers, GeXP simplex PCR detection system was established for six kinds of cattle and sheep viral pathogens respectively. Sensitivity tests of the methods were done and compared with the recommended method of OIE. The results showed that the sensitivity of LSDV and CPDV was 103 copies/μL and the sensitivity of GTPV, PPRV, SPPV and BTV was 102 copies/μL. Visible, GeXP simplex PCR detection method had high sensitivity, it was 10000～100000times higher than the recommended method of OIE.(3) GeXP multiplex PCR detection system was established and optimized for six kinds of cattle and sheep viral pathogens and specificity test and sensitivity test were done. The results of specificity test showed that all of LSDV, CPDV, GTPV, PPRV, SPPV, BTV which had clear, consistent with target fragment size, specific amplification peak were positive. The other virus which didn’t have nonspecific amplification were negative. It’s showed that the established method by this study had good specificity. It’s found that the sensitivity of each of the six pathogens is up to 104copies/μL.(4)Simulated clinical samples were tested by GeXP multiplex PCR and the recommended method of OIE. In all of the 54 simulated clinical samples, the positive rate of GeXP multiplex PCR was94.87%, false positive rate was6.67%, false negative rate was 5.13%. In 35 simulated clinical samples for single infection(except CPDV) and negative control, the positive rate of GeXP multiplex PCR was95%, false positive rate was6.67%, false negative rate was5%;the positive rate of the recommended method of OIE was70%, false positive rate wasO%, false negative rate was30%. The results show that GeXP multiplex PCR method enhanced the positive rate and false negative rate in a large extent, and it could be used for clinical samples for testing.In conclusion, the established GeXP multiplex PCR in this study could simultaneously detect LSDV, CPDV, GTPV, PPRV, SPPV, BTV. This method had good specificity and high sensitivity, providing a fast, accurate and sensitive diagnostic method for the prevention and control of these six kinds of cattle and sheep viral pathogens.