Detection Of Newcastle Disease Virus, Avian Leukosis Virus, Infectious Bronchitis Virus And Infectious Bursal Disease Virus In Wild Birds By Quadruplex RT-PCR

Newcastle diseases virus (NDV), avian leukosis virus (ALV), infectious bronchitis virus (IBV), and infectious bursal diseasevirus (IBDV)are main pathogenic factors of four common avian diaeasewhich leading to great economic losses in poultry farming.For preliminary understanding of the infection status of these four virus in wild birds, a quadruple RT-PCR (quad-RT-PCR)detection method for NDV, ALV, IBV, IBDV detection was established. Based on the highly conserved nucleic acid sequences of virus’ genomes, four sets of universal primers were designed, and four recombinant plasmids containing target sequences were constructed. The reaction system of quad-RT-PCR were optimized by two orthogonal tests, and the specificity and sensitivity were verified. Then, the viscera samples of 67 wild birds were detected by the optimized quad-RT-PCR. RT-PCR results show that target DNA fragments in four different sizes can be amplified:158bp for NDV,131bp for IBV,109bp for ALV and 94bp for IBDV. Specificity tests show that this method can effectively detect these four virus in random virus mixtures and produce negative to Marek’s disease virus, fowl pox virus, egg drop syndrome virus and avian influenza virus. Sensitivity tests show that the minimal detectable limits of NDV, ALV, IBV, IBDV are 102 copy,102 copy,10 copy,10 copy, respectively. According to the actual sample tests, IBDV and NDV can be detected in the wild birds. Conclusion:the optimized quad-RT-PCR can be applied to clinical diagnosis and epidemiological studies of NDV, ALV, IBV, IBDV.