H1N1亚型猪流感病毒3种谱系三重RT-PCR检测方法的建立与应用  

Development and Application of a Triple RT-PCR Assay for Three Main Sublineages of Swine Influenza Virus Subtype H1N1

作　　者：兰德松 魏澍[3,4] 侯振中[1,2] LAN Desong;WEI Su;HOU Zhenzhong(College of Veterinary Medicine,Northeast Agricultural University,Harbin 150030,China;China Ministry of Agriculture Key Laboratory of Animal Pathogen Biology,Harbin 150030,China;Liaoning Provincial Center for Animal Disease Prevention and Control,Shenyang 110164,China;Liaoning Animal Medical Research Institute,Shenyang 110164,China)

机构地区：[1]东北农业大学动物医学院,黑龙江哈尔滨150030 [2]农业部动物疫病病原生物学重点实验室,黑龙江哈尔滨150030 [3]辽宁省动物疫病预防控制中心,辽宁沈阳110164 [4]辽宁省动物医学研究院,辽宁沈阳110164

出　　处：《河南农业科学》2018年第6期122-126,共5页Journal of Henan Agricultural Sciences

基　　金：辽宁省自然科学基金指导计划项目(20170540478);辽宁省科学事业公益研究基金项目(2014001012);辽宁省"百千万人才工程"人选项目(2014921026)

摘　　要：以H1N1亚型猪流感病毒(SIV)3种主要谱系2009甲型H1N1(pdm/09 H1N1)、古典型H1N1(CS H1N1)和类禽型H1N1(a H1N1)的血凝素(Hemagglution,HA)基因为靶基因,设计3对特异性引物,建立一种可以同时检测一个反应体系中H1N1亚型3种主要谱系SIV的三重RT-PCR方法,并将其应用于临床样品的检测,旨在为开展猪群中流行的H1N1亚型SIV感染及流行情况的调查研究提供有力技术支撑。结果显示,该方法可对pdm/09 H1N1、CS H1N1和a H1N1这3个不同谱系SIV特异性检出,扩增片段大小分别为476 bp、293 bp和997 bp,而与其他病毒无交叉反应;最低检测限分别为1 460拷贝/μL、1 700拷贝/μL和1 950拷贝/μL;应用到3 110份临床样品检测中,检测出10株pdm/09 H1N1和21株a H1N1 SIV,未检测到CS H1N1 SIV,与鸡胚分离病毒符合率达100%。综上,该方法敏感性和特异性良好,能快速、有效实现对临床样品中H1N1亚型3种谱系的检测。In order to achieve a quick and efficient detection method for clinical samples,a triplex RT-PCR assay was developed to distinguish the three major sublineages of swine influenza virus(SIV)H1N1 subtype by using three pairs of specific primers derived from the conserved regions of HA gene sequences from 2009 pandemic H1N1(pdm/09 H1N1),classical HIN1(CS H1N1)and avian-like H1N1(aH1N1).The results of the sensitivity test showed that the developed assay was able to specifically amplify three fragments of different lengths,namely 476 bp in pdm/09 H1N1,293 bp in CS HIN1 and 997 bp in aH1N1.There was no cross-reaction with other common viruses.The detection limits were 1 460 copies/μL for pdm/09 H1N1,1 700 copies/μL for CS HIN1 and 1 950 copies/μL for aH1N1 SIV.A total of 3 110 clinical samples were tested by the triplex RT-PCR.The results showed that there were ten samples for pdm/09 H1N1,twenty-one samples for aH1N1,and no sample for CS HIN1,and 100%coincident with culturing-isolation in SPF chicken embryos together with HA and HI test.In conclusion,the triplex RT-PCR was proved to be sensitive and specific for detecting the three major sublineages of SIV H1N1 subtype in clinical samples.

关 键 词：猪流感病毒 2009甲型H1N1 古典型H1N1 类禽型H1N1 三重RT-PCR 

分 类 号：S855.3[农业科学—临床兽医学]