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H3亚型猪流感病毒荧光定量PCR检测方法的建立
引用本文:王云龙,陈小科,韩洁,李玉林,李恒思,王真,邓黎黎.H3亚型猪流感病毒荧光定量PCR检测方法的建立[J].动物医学进展,2009,30(6).
作者姓名:王云龙  陈小科  韩洁  李玉林  李恒思  王真  邓黎黎
作者单位:1. 郑州职业技术学院,河南郑州,450121;河南省生物工程技术研究中心,河南郑州,450001
2. 河南省生物工程技术研究中心,河南郑州,450001
摘    要:通过RT-PCR方法克隆了H3亚型猪流感病毒HA基因一段靶序列,构建重组质粒作为标准阳性模板.根据GenBank中的H3亚型猪流感病毒HA基因保守序列设计了用于FQ-PCR的1对引物和1条TaqMan探针.通过条件优化,以10倍系列稀释的质粒为标准品进行荧光定量PCR扩增,并制作标准曲线,建立了检测H3亚型猪流感的荧光定量PCR方法.结果表明,该方法检测灵敏度可达1.0×100拷贝/μL,线性范围为109~100,达10个数量级;对起始浓度为1.0×109、1.0×108、1.0×107拷贝/μL的标准品的最终实际测得值(Ct)分别为13.68,18.21和20.57;变异系数分别为0.31%、0.17%和0.12%,均小于5%,说明此方法具有良好的准确性和重现性.对阳性组织病料的检测表明,该方法的检测灵敏度高出常规PCR,与套式PCR具有相近的灵敏度.

关 键 词:猪流感病毒  H3亚型  荧光定量PCR  TaqMan探针

Establishment of Fluorescent Quantitative PCR Assay for SIV H3 Serotype Detection
WANG Yun-long,CHEN Xiao-ke,HAN Jie,LI Yu-lin,LI Heng-si,WANG Zhen,DENG Li-li.Establishment of Fluorescent Quantitative PCR Assay for SIV H3 Serotype Detection[J].Progress In Veterinary Medicine,2009,30(6).
Authors:WANG Yun-long  CHEN Xiao-ke  HAN Jie  LI Yu-lin  LI Heng-si  WANG Zhen  DENG Li-li
Institution:WANG Yun-long1,2,CHEN Xiao-ke2,HAN Jie2,LI Yu-lin2,LI Heng-si2,WANG Zhen2,DENG Li-li1,2(1.Zhengzhou Vocational Technical College,Zhengzhou,Henan,450121,China,2.Henan Bioengineering Research Center,450001,China)
Abstract:The part sequences of HA segment of SIV were cloned into pMD-18 T vector and the recombinant plasmid was constructed and used as standard positive template for real time PCR.Meanwhile,according to the published HA segment of SIV sequences of SIV in GenBank,a pair of primers and a TaqMan probe were designed.The FQ-PCR assay was conducted by quantitative concentration of serial 10 fold dilutions of recombinant plasmid DNA by optimizing circulation parameters.A standard curve was achieved and the result showed...
Keywords:SIV  H3 serotype  fluorescent quantitative PCR  TaqMan probe  
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