Quadruplex RT-PCR For Synchronous Detection Of AIV, ALV, IBV And IBDV

Avian influenza virus(AIV), avian leukosis virus(ALV), infectious bronchitis virus(IBV) and infectious bursal disease virus(IBDV) are four important avian pathogens that cause serious economic problems in poultry farming.Development of specific and sensitive techniques for detection of AIV, ALV, IBV&IBDV is very helpful for decrease economic losses caused by these four viral diseases through searching for contagium, clearing away infectious source and blocking route of transmission.Primers for ALV were designed based on conserved sequence of M gene; primers for AIV, IBV&IBDVwere followed related reference. Fragments ofAIV, ALV, IBV&IBDVgenes were amplified from viral RNAs extracted from cultured viruses by unique RT-PCR using specific primers respectively, and were cloned to pMD-18T vector. Then AIV, ALV, IBV&IBDVgenes positive plasmids were successfully constructed.A quadruplex reverse transcription polymerase chain reaction was established based on optimization of annealing temperature, ratio of four kinds of viral specific primers; concentrationof total primers, Taq DNA polymerase, dNTP, Mg2+& PCR buffer. The optimized quadruplex RT-PCR condition is as below:ratio of AIV, ALV, IBV&IBDV specific primers= 2:3:2:3=0.125μM:0.1875μM:0.125μM:0.1875uM,0.075U/μLTaq DNA polymerase,200μMdNTP,2.5mM Mg2+,1.5×PCR Buffer, and 52℃ annealing temperature.Theamplification of 10-fold serial diluted pMD-18T-AIV, pMD-18T-ALV pMD-18T-IBV & pMD-18T-IBDVshows that the detection limit of quadruplex PCR for AIV、ALV、IBV&IBDV is 102copies,103copies,103copies & 103copies respectively. Theamplification of 10-fold serial diluted AIV, ALV, IBV&IBDV reveals that the detection limit of quadruplex RT-PCR for AIV、ALV、IBV&IBDV is 0.0001TCID50, 100fg, 0.00001TCID50&0.001TCID50 respectively.When one kind of, or combination of 2,3 or 4 kinds of pMD-18T-AIV, pMD-18T-ALV, pMD-18T-IBV&pMD-18T-IBDV were used as templates for amplification by quadruplex PCR, one or 2,3 & 4 specific bands were yielded. When cloning plasmids of APV, EDSV, MDV&NDV genes were used as templates, no specific bands were produced. Also when one kind of, or combination of 2,3 or 4 kinds of AIV, ALV, IBV&IBDV were amplified by quadruplex RT-PCR, one or 2,3& 4 specific bands were produced. When APV, EDSV, MDV&NDV were amplified, no specific bands appeared. The results suggested that the quadruplex RT-PCR was not only highly specific, but also could detect out one kind of, or combination of 2,3 or 4 kinds of AIV, ALV, IBV&IBDVsimultaneously.When artificial positive fecal specimen were detected by quadruplex RT-PCR, the assay successfully detected out all one kind of, or combination of 2,3 or 4 kinds of AIV, ALV, IBV&IBDV positive artificial fecal specimen. It suggested that the quadruplex RT-PCR can be used for clinic samples.The quadruplex RT-PCR was used to detected fecal samples of chicken, duck & pigeon collected in Huquan Market of Wuhan City, and tissue specimen of several kinds of wild birds collected in Wuhan City and Daye City. None of 10of chickenfecal samples, 10 duck fecal samples and pigeon fecal samples was positive for AIV, ALV, IBV&IBDV; None of 67 wild birds was positive for AIV, ALV&IBV, but 7 birds were IBDV positive. Sequence analysis of IBDV positive product imply that a new variant may exist in these 7 wild birds.