荧光定量RT-PCR检测H5亚型禽流感病毒方法的建立与验证

›› 2011, Vol. 38 ›› Issue (11): 169-174.

荧光定量RT-PCR检测H5亚型禽流感病毒方法的建立与验证

曹军平^1,2, 陆桂平¹, 徐向明^1,2, 顾敏², 徐全刚², 刘武杰², 彭大新², 刘秀梵²

1. 江苏畜牧兽医职业技术学院,江苏泰州 225300;2. 扬州大学兽医学院,江苏扬州 225009

收稿日期:2011-04-04 修回日期:1900-01-01 出版日期:2011-11-20 发布日期:2011-11-20
通讯作者: 刘秀梵

Development and Validation of Real-time RT-PCR Assay for Detection of Avian Influenza Virus H5 Subtype

CAO Jun-ping^1,2, LU Gui-ping¹, XU Xiang-ming^1,2, GU Min², XU Quan-gang², LIU Wu-jie², PENG Da-xin², LIU Xiu-fan²

1. Jiangsu Vocational and Technical College of Animal Husbandry and Veterinary,Taizhou 225300,China;2. College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China

Received:2011-04-04 Revised:1900-01-01 Online:2011-11-20 Published:2011-11-20

摘要/Abstract

摘要： 根据H5亚型禽流感病毒HA基因上的保守序列,设计合成引物,以H5亚型禽流感病毒HA基因重组质粒为标准品绘制标准曲线,建立了荧光定量逆转录聚合酶链反应检测方法。结果表明,本试验建立的标准曲线循环阈值(Ct值)与模板浓度具有良好的线性关系,相关系数为0.995,灵敏度约为8拷贝/μL,相当于8个AIV颗粒,对新城疫病毒和其他禽病病毒无交叉反应,特异性好、重复性佳,为H5亚型禽流感病毒检测提供了一种特异、敏感、快速、低成本、高通量、生物安全性好的定量检测方法。对178份临床泄殖腔棉拭子样品的检测,该方法结果与经典病毒分离方法符合率大于90.0%,在禽流感病毒临床样品快速筛检、流行病学监测等方面显示了良好的应用前景。

关键词: H5亚型禽流感病毒; 荧光定量RT-PCR; 建立; 验证

Abstract: According to the conservative region of H5 AIV HA gene,the primers were devised and synthesized. A serial 10 fold dilutions positive plasmid was prepared and used for standard. The standard curve revealed the linear relationship between Ct (cycle threshold) and template concentration with a good correlation (R²＝0.995). The RRT-PCR method for the detection of AIV was highly specific and sensitive,and it could be used for rapid quantitative detection of AIV H5 subtype. No cross-reaction was detected against other avian disease viruses. Sensitivity was 8 copies of AIV genome. The total meet rate with traditional virus isolation method was more than 90.0% in detecting 178 clinical cloacal swab samples. It showed that a real-time RT-PCR method for detection of H5 subtype avian influenza virus had been established in this study,which had properties such as high speed,specific,sensitive,cheap,high-throughput,and good biological safety as well as displaying good prospects in the rapid screening of clinical samples and epidemiological monitoring of avian influenza virus H5 subtype.

Key words: AIV H5 subtype; real-time RT-PCR; development; validation

中图分类号:

S858.31

曹军平;陆桂平;徐向明;顾敏;徐全刚;刘武杰;彭大新;刘秀梵. 荧光定量RT-PCR检测H5亚型禽流感病毒方法的建立与验证[J]. , 2011, 38(11): 169-174.

CAO Jun-ping;LU Gui-ping;XU Xiang-ming;GU Min;XU Quan-gang;LIU Wu-jie;PENG Da-xin;LIU Xiu-fan. Development and Validation of Real-time RT-PCR Assay for Detection of Avian Influenza Virus H5 Subtype[J]. , 2011, 38(11): 169-174.

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