禽流感和新城疫病毒二重荧光定量RT-PCR检测方法的建立

目的：建立二重荧光定量RT-PCR方法，用于禽流感病毒（AIV）和新城疫病毒（NDV）的检测。方法：根据AIV和NDV的基因保守序列，设计了AIV和NDV的2对特异性引物和2条用不同荧光基团标记的TaqMan探针；对反应条件和试剂浓度进行优化，建立了能够同时检测AIV和NDV的Z-重荧光定量RT-PCR方法。结果：所建方法特异性好，对AIV和NDV的检测敏感性均达到2000个模板拷贝数，比常规RT-PCR敏感性高100倍；抗干扰能力强，对AIV和NDV不同模板浓度进行组合，仍可有效地同时检测2种病毒。对保存的AIV或NDV鸡胚尿囊液及临床病料进行二重荧光定量RT-PCR检测，结果尿囊液检测的拷贝数均达到10^10/μL以上，临床病料的拷贝数为2．13x10^8-6．52x10^4／μL。结论：建立了用于检测AIV和NDV的二重荧光定量RT-PCR法，该方法特异、敏感、快速、可定量，对AIV和NDV的防制有重要意义。

Development of a Multiplex Real-Time RT-PCR Assay for Detection of Avian Influenza Virus and Newcastle Disease Virus

Objective： To develop a multiplex real-time RT-PCR assay for the identification of avian influenza virus（AIV） and Newcastle disease virus（NDV）. Methods： Two pairs of primer and two TaqMan probes were designed and synthesized according to the conserved gene sequences of AIV and NDV in GenBank. The reaction parameters such as the concentration of two pairs of primers, two TaqMan probes and the reaction buffer were optimized to develop a multiplex real-time RT-PCR assay for the rapid detection of AIV and NDV. Results： The multiplex real-time RT-PCR assay was found to be specific and to be able to detect and differentiate AIV and NDV. The sensitivity of the assay was 2 000 template copies for AIV and NDV, and its sensitivity was 100 times than that of the routine RT-PCR. The samples were examined using the multiplex real-time RT-PCR repeatedly and the results indicated that the multiplex real-time RT-PCR was repro- ducible. Different concentrations of AIV and NDV when mixed together still could be identified by this assay, which implied the assay could be applied to clinical confirmation for simultaneous infection of AIV and NDV. More than 10^10copies/μL of AIV or NDV were detected from allantoic fluid by the multiplex real-time RT-PCR, and 2.13x10S^8-6.52x10^4 copies/μaL of AIV or NDV were detected from clinical samples. Conclusion： The multiplex real-time RT-PCR assay was a quick, sensitive, specific and quantitative tool for the detection of AIV and NDV, and will be useful for the control of avian influenza and Newcastle disease.