基于多重RT-PCR同时鉴定猪流感病毒不同HA和NA基因亚型的DHPLC检测方法的建立  

Establishment of the denaturing high performance liquid chromatography based on multiplex RT-PCR for differentiating HA and NA gene subtypes of swine influenza virus

作　　者：徐超 刘芳[2] 李轲 张巨洲 韩雪清[3] 

机构地区：[1]河南出入境检验检疫局,河南郑州450003 [2]河南农业大学牧医工程学院,河南郑州450001 [3]中国检验检疫科学研究院,北京100029

出　　处：《中国预防兽医学报》2016年第8期624-628,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：质检总局科技计划项目(2013IK304)

摘　　要：为建立快速、敏感并同时鉴定不同HA和NA基因亚型的猪流感病毒(SIV)的方法。本研究根据SIV的H1、H2、H3、H5、H9、N1及N2亚型基因的保守序列分别设计型特异性引物,建立了同时扩增SIV不同亚型的多重RT-PCR及变性高效液相色谱(DHPLC)的高灵敏度和高通量分型检测方法。结果显示,该方法能够特异性的检测并鉴定H1、H2、H3、H5、H9、N1、N2等亚型SIV,而对猪瘟病毒、口蹄疫病毒、猪流行性腹泻病毒、猪繁殖和呼吸综合征病毒均无交叉反应,该方法的最低检出限为100拷贝/μL核酸。对76份猪流感鼻拭子样品临床样品检测结果表明,DHPLC与商业化荧光定量PCR检测试剂盒的检测结果完全一致。本研究建立的方法,特异性强、敏感性高、自动化程度高,为快速分型检测SIV提供技术支撑,并具有良好的应用前景。To establish a rapid, sensitive and simultaneous identification of different subtype swine influenza virus （SIV） HA and NA method, the high sensitivity and high throughput genotyping denaturing high performance liquid chromatography （DHPLC） were developed based on multiplex RT-PCR amplification with the subtype-specific primers designed according to conserved sequences of SIV HI, H2, H3, H5, H9, N1 and N2 subtype genes. The results show that this method was capable to detect and identify specific HI, H2, H3, H5, H9, N1, N2 subtypes of SIV, but had no cross-reaction with classical swine fever virus, foot and mouth disease virus, porcine epidemic diarrhea virus and porcine reproductive and respiratory syndrome virus. The detection limit was 100 copies/μL of the SIV. A total of 76 clinical samples of SIV nasal swab were tested, the results showed the correlation between the DHPLC and commercialized real-time PCR kit was 100%. The established specificity, high sensitivity and automatic method provided the technical support for the rapid genotyping of SIV.

关 键 词：猪流感病毒 亚型 变性高效液相色谱技术 分型检测 

分 类 号：S852.65[农业科学—基础兽医学]