Development and Application of a Triple RT-qPCR Assay for the Differential Detection of Newcastle Disease Virus,Avian Infectious Bronchitis Virus and Avian Influenza Virus
Development and Application of a Triple RT-qPCR Assay for the Differential Detection of Newcastle Disease Virus,Avian Infectious Bronchitis Virus and Avian Influenza Virus
Newcastle disease,avian infectious bronchitis and avian influenza are acute infectious diseases that all cause respiratory symptoms in birds,they have similar symptoms and mixed infections may occur.In order to develop a triple fluorescent RT-qPCR method for the detection of NDV,IBV and AI viruses,the present study compared the genes of NDV L gene,IBV 1a gene and AIV NP gene registered in GenBank,and designed three sets of primers and probes by selecting conserved sequences through analysis to optimize the reaction system of the detection method.Ultimately,a triple RT-qPCR method for simultaneous detection of NDV,IBV and AIV was developed successfully,and its specificity,sensitivity and reproducibility were evaluated.The results showed that the method was able to specifically detect NDV,IBV and AIV with good specificity;the lower detection limit of the method was 3.02×102copies/μL for all the three viruses,indicating a high sensitivity;and the results of the reproducibility experiments showed that the coefficients of variation of intra-and inter-batch were no more than 1.28%,showing an excellent.One hundred and two clinical samples were tested using the method established in this study and the results were compared with those obtained by conventional RT-PCR with an overall agreement rate of 96.6%.The triple RT-qPCR assay established in this study is specific and sensitive,and can be used for the detection of the three viruses:NDV,IBV,and AIV.
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