Development And Application Of The Multiplex PCR Assay For Duck Tembusu,Bronchitis And Newcastle Disease Viruses

Tembusu Virus(TMUV)was an infectious pathogen of Duck,which caused duck egg drop,growth retardation,and even death of reported in April 2010.In recent years,people also found that the virus could infect chickens,causing a loss to poultry industry.TMUV had many similaritiesin symptoms with other respiratory pathogens,including new castle disease virus(NDV),infectious bronchitis virus(IBV)and avian influenza virus(AIV).It has appeared mixed infections between TMUV and other virusin Shanghai.Depending on clinical symptoms could not performthe differential diagnosis to these multiple infection of pathogens.In addition,Chicken populations often have the same individuals at the same time by different viral infections.Therefore,if we can simultaneously detect the same disease in different types of viruses,the epidemiological investigation and the differential diagnosis of disease play a very important role.In this study,we have established a multiple PCR detection method of the three kinds of avian viruses(NDV,IBV,TMUV).Through the analysis of bioinformatics,three kinds of specific primers of virus were designed.The concentration of primers and PCR reaction annealing temperature gradient had been performed optimization.The results showed that primer concentration in 0.1 ~ 10 pmol/mL and annealing temperature in 53 ? or 56 ?,can amplify the complete virus genes,386 bp(IBV),588 bp(NDV)and 821 bp(TMUV)respectively.The method of multiple PCR could accurately amplify out three kinds of pathogens(NDV,IBV and TMUV)of segments of nucleic acid,without amplifying of the other pathogens.The method of of multiple PCR showed a strong specificity.Meanwhile,the method of multiple PCR could simultaneously detect 10 ng of NDV,100 pg of IBV and 100 pg of TMUV,which showed a high sensicitivity.In addition,the technology of multiple PCR also could detect NDV,IBV,TMUV the infection alone and the mixed infection of NDV,IBV and TMUV which showed clinical application.In conclusion,our study successfully established the multiple PCR detection of three methods of avian virus,which contributed to provide technical support of the effective prevention,control and eradicationof fowl disease.