Establishment and application of a multiplex PCR assay for simultaneous detection of AIV,NDV,IBV,ILTV and MG

According to the gene sequences of avian influenza virus(AIV),Newcastle disease virus(NDV),infectious bronchitis virus(IBV),infectious laryngotracheitis virus(ILTV) and Mycoplasma gallisepticum(MG) available in GenBank,five pairs of specific primers were designed and synthesized to develop a multiplex PCR for simultaneous detection of AIV,NDV,IBV,ILTV and MG.The annealing temperature,primer concentration and enzyme concentration in the multiplex PCR were optimized and the specificity and sensitivity of the multiplex PCR were tested and some clinical samples were detected by the multiplex PCR.In result,the gene fragments of approximate 268 bp(AIV),312 bp(NDV),457 bp(IBV),662 bp(ILTV) and 732 bp(MG) were simultaneously amplified from the same sample containing the RNAs of AIV,NDV and IBV and the DNAs of ILTV and MG.The specificity and sensitivity tests showed that as little as 1 pg RNA of AIV,1 pg RNA of NDV,10 pg RNA of IBV,1 pg DNA of ILTV and 10 pg DNA of MG could be detected by this multiplex PCR.The clinical samples detections showed that the developed multiplex PCR could be widely used to diagnose simultaneously the five pathogens of AIV,NDV,IBV,ILTV and MG in Chicken.