Development And Application Of Duck Susceptible Virus Detection Method

The Food and Agriculture Organization(FAO)suggests that approximately 739 million ducks are raised annually in China,accounting for 63.76%of the World's stock in 2016.However,along with the development of China's duck industry,viral infection has been more and more serious because of the excessive stocking,lack of effective management,enhanced mobility of humans and animals and environment pollution.The major viruses that cause enormous economic losses to duck industry include avian influenza virus(AIV),Newcastle disease virus(NDV),goose parvovirus(GPV),duck hepatitis A virus-1(DHAV-1),duck Tembusu virus(DTMUV),fowl adenovirus-4(FAdV-4)and duck enteritis virus(DEV).In this research,the above common viruses were introduced in terms of pathogens,epidemiology,clinical symptoms and pathological changes;besides,the advantages and disadvantages of common detection methods for these viruses were compared.It is essential to develope PCR detection methods based on nucleic acid detection technology,which can be used to detect duck viruses clinically.Experiment 1.Establishment of uniplex PCR method for detection of seven duck virusesIn this experiment,the sequences of seven viruses that were susceptible to ducks were downloaded and aligned.Then seven pairs of specific primers were designed in the conserved regions of the conserved genes.We verified its specificity,sensitivity and repeatability.The results showed that the method had strong specificity and good repeatability.The detection limits of FAdV and DHAV were 1×103 copies/?L;the detection limit of AIV was 1×101 copies/?L;the detection limits of DEV,DTMUV,NDV and GPV were 1×102 copies/?L.Experiment 2.Establishment of multiplex PCR method for simultaneous detection of seven duck virusesThe multiplex PCR(m-PCR)method was established on the base of uniplex PCR.The optimum parameters for m-PCR were annealing temperature at 57?,Mg2+ concentration at 4 mM,Taq DNA polymerase concentration at 0.05 U/?L,and dNTP concentration at 0.32 mM.With these optimal parameters,the m-PCR method produced neither cross-reactions among these seven viruses nor nonspecific reactions with other common waterfowl pathogens.The detection limit of m-PCR for each virus was 1×104 copies/?L.In addition,the m-PCR method could detect a combination of several random viruses in co-infection analysis.Finally,the m-PCR method was successfully applied to clinical samples,and the detection results were consistent with uniplex PCR.Experiment 3.Establishment of multiplex real-time PCR method for simultaneous detection of three duck virusesThis experiment also developed multiple real-time PCR method to detect three duck viruses simultaneously.The results showed that the multiplex quantitative PCR method had high specificity;the detection limits of AIV and NDV were 1×103 copies/?L,the detection limit of DTMUV was 1 ×102 copies/?L;the intra-assay CV value was less than 2%,and the inter-assay CV value was less than 5%,which had good repeatability.