鸡传染性喉气管炎病毒PCR检测方式的建立和应用  

Establishment and Application of Polymerase Chain Reaction for Infectious Laryngotracheitis Virus

作　　者：李海利[1] 易秀玲 郎利敏[1] 徐引弟[1] 焦文强[1] 张立宪[1] 张青娴[1] 游一[1] 王克领[1] 李奎[2] 

机构地区：[1]河南省农业科学院畜牧兽医研究所,河南郑州450002 [2]河南农业大学牧医工程学院,河南郑州450002

出　　处：《现代畜牧兽医》2016年第3期1-7,共7页Modern Journal of Animal Husbandry and Veterinary Medicine

基　　金：河南省农业科学院自主创新基金

摘　　要：根据GenBank传染性喉气管炎病毒(ILTV)基因序列,设计合成了1对特异性引物。以ILTV DNA为模板,建立了检测ILTV的PCR检测方法。通过优化PCR反应条件,成功扩增出一条约690 bp的目的基因片段。而鸡传染性法氏囊病病毒、新城疫病毒、传染性支气管炎病毒、禽流感病毒基因组均未扩增为出相应的片段。敏感性试验检测出其DNA最小检出量为10-2μg。重复性试验中对3份检测为传染性喉气管炎病毒阳性病料进行检测,发现3次重复检测的结果完全一致。临床应用中运用上述优化的PCR反应条件进行PCR检测临床送检的16份疑似鸡传染性喉气管炎病毒感染病料,结果显示检出阳性样品6份,将阳性样品的PCR扩增产物克隆后序列分析显示均为鸡传染性喉气管炎病毒。结果表明,建立的PCR方法具有良好的特异性,敏感性和稳定性,可应用于传染性喉气管炎病毒鉴定和临床诊断。According to the gene sequence of Infectious Laryngotracheitis Virus （ILTV） that published in GenBank, the experiment designed and synthesized a pair of specific primers. Using the genomic DNA of ILTV as a template, a PCR method was established for the detection of ILTV gene. By optimizing PCR reaction conditions, a 690 bp fragment was amplified from the genome of ILTV, but not from IBDV, NDV , IBV and AIV. The sensitivity test showed that the minimum detectable amount of its DNA was 10^2 μg. The Repeatability test detected 3 copies of testing positive for Infectious Laryngotracheitis Virus and showed the same results. Using the optimized PCR reaction conditions in the clinical application for detecting 16 samples which were Suspected that infected with Infectious Laryngotracheitis Virus, the testing results showed 6 positive samples. Aftering cloning and analyzing sequence of the positive samples＇ PCR amplification product, it showed that all infected with Infectious Laryngotracheitis Virus. The results indicated that the PCR method has good specificity, sensitivity and stability, and can be used for the detection of ILTV.

关 键 词：鸡传染性喉气管炎病毒 PCR 检测 

分 类 号：S852.65[农业科学—基础兽医学]