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Establishment and application of multiplex PCR assay for detecting avian influenza virus,newcastle disease virus and angara disease virus

ZHANG Man;HAN Fei;College of Animal Engineering,Yangling Vocational&Technical College;  
【Objective】A multiplex reverse transcription-polymerase chain reaction(PCR)assay was developed for the detection of Avian influenza virus(AIV),Newcastle disease virus(NDV)and Angara disease virus(FAV-4).【Method】According to the M genome sequence of AIV,F genome sequence of NDV and Hexongenome sequence of FAV-4 in Genbank,three specific pairs of primers were synthetized.The DNA/RNA of these three virus were extracted.The primer concentrations and annealing temperature of multiplex PCR reaction condition were optimized and the multiplex PCR for simultaneously detecting AIV,NDV,and FAV-4 was established.The specificity,sensitivity and repeatability of the multiplex PCR were detected.The multiplex PCR method was applied to detect 67 clinical samples,and the results were compared to single PCR detection.【Result】The multiplex PCR for simultaneously detecting AIV,NDV,and FAV-4 was established successfully.The method has high sensitivity and specificity.No amplification was accepted from IBV,IBDV,FPV,and MDV.The multiplex PCR could detect 24.3 pg/μL of NDV nucle-ic acid,30.4 pg/μL of FAV-4 nucleic acid and 28.0 pg/μL of AIV nucleic acid.The results of multiplex PCR on testing clinical samples were consistent with the established PCR detection method.【Conclusion】The multiplex PCR for detecting AIV,NDV,and FAV-4 can be used in clinic.
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