Establishment Of A Multiplex Suspension Array For Detection Of Five Exotic Pathogens

The spread of exotic pathogens always causes serious economic losses,thus directly threatens the economic development of the country and social harmony and stability.In recent years,with the increasing frequence of import and export trade in our country,the risk of importing exotic pathogens has also increased.Therefore,the quarantine of exotic pathogens is getting more and more attention.African Swine Fever(ASF),Peste Des Petits Ruminants(PPR),West Nile Fever(WNF),Rift Valley Fever(RVF)and Southen African Territories Foot-and-Mouth Disease(SAT FMD)are five exotic animal diseases that have been specifically pointed out by our country which must prevent intensively.So,it is of great significance to establish a rapid and accurate method for detection of these five exotic pathogens.According to the published p72 gene sequence of ASFV,N gene sequence of PPRV,E gene sequence of WNV,segment S gene sequence of RVFV and 5’UTR gene sequence of SAT FMDV(SAT1,SAT2,SAT3)in GenBank,a comparative analysis was performed to select highly conservative sequences of each viral gene.Then we designed specific primers and probes of each virus and coupled labeled probes with fluorescent microspheres.Next,target fragments amplified by asymmetric PCR were hybridized with C12-modified probe-coupled fluorescent microspheres,and fluorescent signals were read by the Bio-plex system.Finally,results were analyzed according to qualitative criteria.Based on the optimization of reaction conditions such as the ratio of primers for asymmetric PCR,hybridization temperature and hybridization time.The simplex suspension array for detection of ASFV,PPRV,WNV,RVFV and SAT FMDV was established successfully.The results showed good sensitivity and specificity and there have no cross reaction between these five virus in this method.Based on this,a multiplex suspension array for detection of ASFV,PPRV,WNV,RVFV and SAT FMDV has been established.Using this multiplex suspension array method to test 106 samples compared to conventional PCR methods.The results were 100%consistent.In this study,we successfully established single and multiplex suspension array to detect ASFV,PPRV,WNV,RVFV and SAT FMDV successfully.Thus,a new method for rapid,high-throughput detection of exotic pathogens has been established and it also provided a new idea for detection of other viral pathogens.