H5亚型禽流感病毒环介导等温扩增检测方法的建立  

Detection of H5 Avian influenza virus by Loop-mediated Isothermal Amplification

作　　者：黄书林[1] 吴彤[2] 蒋菲[1] 闫新博 吴文学[1] 

机构地区：[1]中国农业大学动物医学院,北京100193 [2]南京农业大学生命科学学院,南京210095

出　　处：《农业生物技术学报》2016年第9期1384-1391,共8页Journal of Agricultural Biotechnology

基　　金：国家公益性行业(农业)科研专项(No.201203056)

摘　　要：本研究利用反转录环介导等温扩增(reverse transcription loop-mediated isothermal amplification,RT-LAMP)技术建立了H5亚型禽流感病毒(Avian influenza virus,AIV)检测方法。首先,针对H5亚型禽流感病毒血凝素(hemagglutinin,HA)基因8个保守区域设计了3对扩增引物。然后,添加到RT-LAMP反应体系中,并加入优选的冻干保护剂进行冷冻干燥。取等温扩增冻干试剂,用双蒸水溶解并恢复至原体积后,在63℃反应1 h,可检出102拷贝的目的基因,比RT-PCR方法敏感约10倍,并且证明冷冻干燥对等温扩增反应敏感性没有影响。另外,利用等温扩增冻干试剂对禽流感病毒、新城疫病毒(Newcastle disease virus,NDV)和传染性支气管炎病毒(Infectious bronchitis virus,IBV)进行检测,结果显示特异性良好。为了便于临床应用,在反应结束后加入由SYBR Green I与羟基萘酚蓝(hydroxynaphthol blue,HNB)组成的显色液,可通过肉眼观察颜色变化判定RT-LAMP结果,与琼脂糖凝胶电泳方法判定结果一致。为考察等温扩增冻干试剂的临床应用效果,对326份临床样品进行了检测。结果表明,RT-LAMP与RT-PCR检出阳性样本数分别为38份和31份,表明RT-LAMP方法阳性检出率高于RT-PCR方法。因此,本研究建立的H5亚型流感病毒RT-LAMP反应体系,可以冻干处理并经显色观察结果,便于研制成试剂盒产品,其敏感性高、特异性好、操作简捷,利于在基层兽医实验室和养殖场推广应用。Influenza in birds is caused by infection with viruses of the family Orthomyxoviridae placed in the genus influenza virus A. Many species of birds have been shown to be susceptible to influenza A viruses. Influenza A viruses are classified into subtypes on the basis of their haemagglutinin （HA） and neuraminidase （NA） antigens. Traditional diagnostic methods of the subtype H5 influenza are laborious, time-consuming or insensitive, such as virus isolation, haemagglutination, neuraminidase inhibition and RT-PCR. So, more sensitive and convenient methods are in demand. Reverse transcription loop-mediated isothermal amplification （RT-LAMP） offers the advantage of omitting thermocycling, enabling RNA amplification at constant temperature, and is used most widely among the isothermal amplification technologies. In order to develop a detection method of subtype H5 Avian influenza virus （AIV） with RT-LAMP, 3 pairs of primers specifically directing to 8 recognition sites on the hemagglutinin （HA） gene of H5-AIV were designed, and then mixed into the other ingredients as recommended in the instruction of the RT-LAMP kits with or without freeze-dried protecting agents. The recombinant plasmid with the whole genome of subtype H5N1-AIV was extracted, purified and diluted to make each reaction system of RT-LAMP and RT-PCR contain 10^6, 10^5, 10^4, 10^3, 10^2, 10^1 or 10^0 copies. The sensitivities of the RT-LAMP before and after lyophilization, and the difference on sensitivity between RT-LAMP and RT-PCR by tm＇bidity monitoring and agarose gel electrophoresis analysis, respectively, were determined and compared. The specificities of RT-LAMP and RT-PCR with subtypes H1-H15 AIV, Newcastle disease virus（NDV） and Infectious bronchitis virus （IBV） were tested. The feasibility of the visualization of RT- LAMP was tested by adding the color developing solution （mixture of SYBR Green I and hydroxynaphthol blue） into the isothermal amplification systems after reaction for 1 h at 63 ℃. Finally, totally

关 键 词：反转录环介导等温扩增(RT-LAMP) H5亚型禽流感病毒(H5-AIV) 冻干 RT-PCR 显色 

分 类 号：S855.3[农业科学—临床兽医学]