Development Of A Multiplex Liquid Chip Assay For Detection Of Multiple Exotic Diseases Of Swine

Pig borne exogenous diseases can be introduced into China through international trade,wildlife carrying and insect vectors.Once introduced into China,it will bring serious losses to the pig industry,even public health problems.In this study,African Swine Fever Virus(ASFV),Nipahvirus(NIV),Vesicular Stomatitis Virus(VSV),Seneca Valley Virus(SVV)and Foot-and-Mouth disease Virus(FMDV)were used as the research object.The specific primers were designed,and the modified products were obtained by multiplex PCR.Then,they were hybridized and incubated with microspheres,and the sensitivity and specificity of the method were verified,in order to construct a liquid-phase chip detection system for a variety of swine exotic diseases.The main results are as follows:1.Establishment of a single PCR system for each exotic pig diseasesThe results showed that the lowest sensitivity of single PCR for ASFV,NIV,VSV,SVV and FMDV was 39.0 copies/μL,4.6 copies/μL,29.9 copies/μL,36.8 copies/μL and 20.9 copies/p L,respectively.2.Establishment of multiplex PCR system for multiple pig derived alien diseasesOn the basis of single PCR,the annealing temperature of multiple PCR was optimized to determine the optimal reaction conditions and detection system.The detection limit of ASFV,NIV,VSV,SVV and FMDV were 3.90x102copies/μL,4.60x104copies/μL,2.99x104copies/μL,3.68x102copies/μL and 2.09x102copies/μL,respectively.The detection limit of ASFV was 3.90x103copies/μL,SVV was 3.68x104copies/μL,FMDV was 2.09x103copies/μL and NIV were respectively VSV is not easy to distinguish from gel electrophoresis.The results of specific experiments show that the multiplex PCR method just amplify specific bands.3.The establishment of liquid chip detection method for multiple pig exogenous diseasesThe PCR products were hybridized with microspheres,and then incubated with streptavidin phycoerythrin.After incubation,Luminex 200 liquid chip was used to detect the fluorescence median.The optimum crossing conditions were determined by optimizing the crossing time and temperature.MFI value of all positive results more than 3 times of negative control,and negative control less than 300.In the sensitivity test results of ASFV,NIV,VSV,SVV and FMDV,the detection limit of single plasmid liquid chip was 39.0copies/μL,4.60x104copies/μL,2.99x103copies/μL,36.8copies/μL and 2.09x102copies/μ L,respectively;the lowest detection quantity of mixed plasmid liquid chip was 3.90x102copies/μL,4.60x104copies/μL,2.99x105copies/μL,3.68x103copies/μL and 2.09 x102copies/μL。The liquid chip detection method only reacted specifically to the five viruses studied,and had no cross reaction with other control viruses.The coefficient of variation of three groups in the same batch was less than 0.2%.In this experiment,the liquid chip detection methods of ASFV,NIV,VSV,SVV and FMDV were successfully established,which provided technical support for the rapid and high-throughput detection of porcine viral diseases,and also provided ideas for the establishment of liquid chip detection methods of other pathogens.