鸡传染性支气管炎病毒H52株TaqMan-MGB 荧光定量RT-PCR检测方法的建立

›› 2012, Vol. 39 ›› Issue (4): 169-173.

鸡传染性支气管炎病毒H52株TaqMan-MGB 荧光定量RT-PCR检测方法的建立

梁耀峰^1,2, 郭霄峰¹, 宋立², 刘洋^2,3

1. 华南农业大学兽医学院,广东广州 510642;2. 中国兽医药品监察所,北京 100081;3. 扬州大学兽医学院,江苏扬州 225009

收稿日期:2011-10-28 修回日期:1900-01-01 出版日期:2012-04-20 发布日期:2012-04-20
通讯作者: 宋立

Establishment of TaqMan-MGB Fluorescence Quantitative RT-PCR Assay for Detection of Avian Infectious Bronchitis Virus H52 Strain

LIANG Yao-feng^1,2, GUO Xiao-feng¹, SONG Li², LIU Yang^2,3

1. College of Veterinary Medicine,South China Agricultural University,Guangzhou 510642,China;2. China Institute of Veterinary Drug Control,Beijing 100081,China;3. College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China

Received:2011-10-28 Revised:1900-01-01 Online:2012-04-20 Published:2012-04-20

摘要/Abstract

摘要： 本研究建立了一种快速的鸡传染性支气管炎病毒(IBV)H52株定量检测方法,为进一步研究疫苗的效价检测和质量监控新方法奠定基础。针对IBV核蛋白(N)基因,设计特异的H52株荧光定量PCR检测引物和TaqMan-MGB探针,优化反应体系和条件后进行特异性、敏感性、重复性试验,探讨荧光定量RT-PCR与EID₅₀方法测定病毒含量的相关性,并通过体外转录技术对病毒基因拷贝数进行定量分析。该方法特异性强,与其他禽源病毒均无交叉反应;该方法可检测到5.60×10¹拷贝/μL的病毒核酸,与常规PCR相比,敏感性高10倍;重复性试验的变异系数为0.9%~3.2%;与EID₅₀检测结果的相关系数r＝0.934,两种方法在检测H52株病毒效价上具有很好的相关性。本研究建立的TaqMan-MGB荧光定量RT-PCR方法,适合于对鸡传染性支气管炎病毒H52株病毒含量的快速定量检测。

关键词: 荧光定量RT-PCR; TaqMan-MGB探针; 鸡传染性支气管炎病毒; H52

Abstract: A rapid assay for detecting avian infectious bronchitis virus was established and laid the foundation for further study on new methods of vaccine potency testing and quality control. Primers and probe of fluorescence quantitative PCR were designed for H52 strain, according to the N gene of IBV. Then the method was optimized. Specificity, sensitivity, and repeatability were tested and the correlation was explored between the quantitative RT-PCR method and EID₅₀ on the determination of virus quntity. In addition, in vitro transcription technology was used to develop a quantitative PCR format with virus copies amount. It was highly specific and no cross-reactions were found with other avian pathogens. The assay had a detection limit of 5.60×10¹copies/μL viral RNA, which was 10 times more sensitive than the conventional PCR. Coefficient of variation value ranged from 0.9% to 3.2% in repeatability test. Compared with EID₅₀ test results, the correlation coefficient r was 0.934, which showed that two methods in detecting the virus titer on the H52 strain had a good correlation. This assay of TaqMan-MGB fluorescence quantitative RT-PCR is suitable for rapid and quantitative detection of IBV H52 strain.

Key words: luorescence quantitative RT-PCR; TaqMan-MGB probe; avian infectious bronchitis virus; H52

中图分类号:

S858.31

梁耀峰;郭霄峰;宋立;刘洋;. 鸡传染性支气管炎病毒H52株TaqMan-MGB 荧光定量RT-PCR检测方法的建立[J]. , 2012, 39(4): 169-173.

LIANG Yao-feng;GUO Xiao-feng;SONG Li;LIU Yang;. Establishment of TaqMan-MGB Fluorescence Quantitative RT-PCR Assay for Detection of Avian Infectious Bronchitis Virus H52 Strain[J]. , 2012, 39(4): 169-173.

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