H7N9亚型禽流感病毒HRM检测方法的建立  

Development of HRM method for detection of H7N9 subtype avian influenza virus

作　　者：刘志成[1] 贾伟新[2] 孙敏华[1] 沈海燕[1] 孙俊颖[1] 殷三鸿[3] 钟亮宁[3] 张建峰[1] 张春红[1] LIU Zhi-cheng JIA Wei-xin SUN Min-hua SHEN Hai-yan SUN Jun-ying YIN San-hong ZHONG Liang-ning ZHANG Jian-feng ZHANG Chun-hong(Guangdong Open Laboratory of Veterinary Public Health/Guangdong Key Research Laboratory of Livestock Disease Prevention/Institute of Animal Health, Guangdong Academy of A gricultural Sciences, Guangzhou 510640, China National and Regional Joint Engineering Laboratory for Medicament of Zoonosis Prevention and Control/College of Veterinary Medicine, South China A gricultural University, Guangzhou 510fi42, China Center for Animal Disease Control and Prevention of Dongguan Prefecture ,Dongguan 523086, China)

机构地区：[1]广东省农业科学院动物卫生研究所广东省畜禽疫病防治研究重点实验室广东省兽医公共卫生公共实验室,广东广州510640 [2]华南农业大学兽医学院人兽共患病防控制剂国家地方联合工程实验室,广东广州510642 [3]东莞市动物疫病预防控制中心,广东东莞523086

出　　处：《中国兽医科学》2016年第11期1388-1393,共6页Chinese Veterinary Science

基　　金：国家科技部科研计划项目(YS2016YFNC020088;2014DFA31730);广东省科技计划项目(2013B020224004;2015B0-50501007;2015B070701015;2014A040401048);广东省农业厅专项项目(粤农函[2014]1046号);广州市科技计划项目(201508020055);东莞市科技计划项目(2014108101041)

摘　　要：根据H 7N 9亚型禽流感病毒(AIV)的HA和NA基因保守区域,分别设计了2对特异性引物,通过优化反应条件,建立了基于高分辨率熔解曲线(HRM)分析的H7N9亚型AIV检测方法。结果显示,该方法特异性强,其他常见禽源病毒及非H7亚型和非N9亚型的AIV不能形成特异熔解峰形;灵敏度高,对H7和N9阳性质粒的最低检测限分别达到1.0×101 copies/L和1.0×100 copies/L;重复性好,熔解峰Tm 1和Tm2的批内和批间变异系数均<1%;临床样本检测与某商品化H7N9亚型AIV定量PCR检测试剂盒比较,符合率为100%。该方法可用于临床疑似病例的应急检测、早期诊断及养鸡场、活禽交易市场等外环境H7N9亚型AIV的监测。Two pairs of primers were designed according to the conserved sequences of the hemagglu- tinin （HA） genes and the neuramidinase （NA） genes of H7N9 subtype avian influenza virus （AIV）,respec- tively. An optimized HRMmethod to rapidly detect the H7N9 subtype AIV was developed and evaluated, lt was shown that the specificity of this method was high,the melting curve was displayed for the detec- tion of H7 subtype and N9 subtype and HTN9 subtype AIV,and no positive results were observed when nu- cleic acid from other subtypes AIV and avian pathogens;the detection limit of this method was as low as 1.0×10^1copies/μL for the positive plasmids p-H7 and 1.0 ×10^0 copies/μL for p-N9,respectively. The variations of both inter-and intra-assay of the peak melting temperatures Tm1andTm2were less than i%. Moreover,compared with the results of a commercial real-time PCR kit for clinical samples,the HRMmethod showed the coincidence rate was 100%.The method can be used for emergency detection and early diagnosis of clinical suspected avian with H7N9 subtype AIV infection,and for surveillance of the virus in external environments like chicken farms and live poultry markets.

关 键 词：禽流感 H7N9 高分辨率熔解曲线 

分 类 号：S852.659.5[农业科学—基础兽医学]