| 摘要: |
| 【目的】建立鉴别禽流感病毒（AIV）、新城疫病毒（NDV）和安卡拉病毒（FAV-4）的多重PCR检测方法。【方法】根据GenBank已登录的AIV的M基因、NDV的F基因和FAV-4的Hexon基因序列，设计合成了3对特异性引物。提取3种病毒DNA/RNA，通过优化多重PCR反应中的引物浓度和退火温度，建立快速鉴别3种病毒的多重PCR方法，并对该方法的特异性、敏感性和重复性进行检验。应用建立的多重PCR方法对67份临床病料进行检测，并将其结果与已建立的单重PCR检测结果进行比较。【结果】成功建立了能快速鉴别3种病毒的多重PCR方法，该方法特异性良好，对传染性支气管炎病毒(IBV)、传染性法氏囊病病毒(IBDV)、鸡痘病毒(FPV)、鸡马立克病病毒(MDV)的扩增结果均为阴性；敏感性较强，能检测出的NDV、FAV-4和AIV最低核酸质量浓度分别为24.3，30.4和28.0 pg/μL。多重PCR方法临床病料的检测结果与单重PCR检测结果一致。【结论】建立的AIV、NDV和FAV-4多重PCR检测方法方法可以用于临床上3种病原的鉴别诊断。 |
| 关键词: 多重PCR 禽流感病毒 新城疫病毒 安卡拉病毒 |
| DOI： |
| 分类号: |
| 基金项目:陕西省科技攻关项目（2016NY-096）；陕西杨凌农业高新技术示范区重大科技项目（2016-XY-16） |
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| Establishment and application of multiplex PCR assay for detecting avian influenza virus,newcastle disease virus and angara disease virus |
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ZHANG Man,HAN Fei
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| Abstract: |
| 【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 Hexon genome 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 nucleic 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. |
| Key words: multiplex PCR avian influenza newcastle disease angrara disease |
