一步法多重RT-PCR检测新城疫病毒、H9亚型禽流感病毒和传染性法氏囊病病毒方法的建立和应用

发布时间：2018-06-19 03:01

本文选题：新城疫病毒 + H亚型禽流感病毒　；参考：《养禽与禽病防治》2016年04期

【摘要】：根椐GenBank中登录的新城疫病毒(NDV)、H9亚型禽流感病毒[AIV(H9)]和传染性法氏囊病病毒(IBDV)基因序列,分别设计3对引物,在建立各病毒单项一步法RT-PCR技术的基础上,优化多重RT-PCR反应条件,建立了3种病毒的一步法多重RT-PCR检测方法,用这3对引物对同一样品中的NDV、AIV(H9)和IBDV核酸模板进行多重RT-PCR扩增,结果可同时扩增NDV的466bp、AIV(H9)的685bp和IBDV的244bp的特异性片段,而对其他4种病原的扩增结果均为阴性。敏感性测定结果表明,该多重RT-PCR技术能检出10pg的NDV、10pg的AIV(H9)和1pg的IBDV模板。用53份临床病料对本研究多重RT-PCR技术和单项RT-PCR技术进行对比验证,结果显示,两者的总符合率为100%。表明建立的一步法多重RT-PCR检测方法,具有特异、快速及准确的特点,可用于对这3种病毒的同时检测和鉴别诊断。
[Abstract]:Three pairs of primers were designed based on the sequence of NDV / H9 subtype avian influenza virus (NDV) and infectious bursal disease virus (IBDVV) gene of Newcastle disease virus (NDV) and Infectious bursal Disease virus (IBDVV) registered in GenBank. The conditions of multiplex RT-PCR were optimized on the basis of single one-step RT-PCR of each virus. A one step multiplex RT-PCR method was established for the detection of three viruses. The three pairs of primers were used to amplify NDV AIVH9) and IBDV nucleic acid template in the same sample. The results showed that the specific fragments of 685bp and IBDV 244bp of NDV 466bpAIVAIVH9) and IBDV could be amplified simultaneously. The amplification results of the other four pathogens were all negative. The sensitivity test showed that the multiplex RT-PCR could detect the 10pg NDV 10pg AIVDV 9) and the 1pg IBDV template. The multiplex RT-PCR technique and single RT-PCR technique were compared with 53 clinical samples. The results showed that the total coincidence rate of the two methods was 100. The results showed that the one-step multiplex RT-PCR assay was specific, rapid and accurate, and could be used for simultaneous detection and differential diagnosis of the three viruses.
【作者单位】：山东绿都生物科技有限公司;山东省滨州畜牧兽医研究院;
【基金】：山东省现代产业技术体系家禽创新团队生产与环境控制创新团队项目(SDAIT-13-011-10) 山东省重点研发计划项目(2015GSF121027) 2015年度山东省引进国外智力成果示范推广项目
【分类号】：S852.65

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