坦布苏病毒荧光定量RT-PCR方法的建立

doi:10.3864/j.issn.0578-1752.2012.21.018

摘要/Abstract

摘要： 【目的】利用SYBR GreenⅠ荧光定量技术建立一种相对定量检测坦布苏病毒的方法。【方法】针对坦布苏病毒NS5、E基因分别设计了1对特异性引物，同时设计1对扩增内参基因β-actin引物，将PCR扩增的片段分别连接到pMD18-T载体上构建重组质粒，经筛选、鉴定纯化后，倍比稀释作为质控样品，用于实时荧光定量PCR中NS5、E基因及内参基因β-actin标准曲线的构建，并进行反应的灵敏性、特异性和重复性试验。【结果】结果显示标准曲线线性关系R2值均在0.99 以上, 检测极限约为1.0E+01拷贝数质粒DNA；特异性结果表明只能检测到坦布苏病毒的扩增曲线；批内和批间重复性试验的变异系数均小于0.5%；用已建立的方法对临床样品进行3次重复检测，病毒RNA的检出率为100%。【结论】本研究初步建立了基于坦布苏病毒NS5、E基因的SYBR GreenⅠ荧光定量RT-PCR的方法，为养鸭场诊断和监测坦布苏病毒提供了一种新的特异、灵敏的检测方法。

关键词: 坦布苏病毒, NS5基因, E基因, SYBR GreenⅠ实时荧光定量RT-PCR, 相对定量

Abstract: 【Objective】The objective of the study is to establish a method for detecting Tembusu virus by SYBR GreenⅠ relative fluorescence quantitative RT-PCR. 【Method】 Special primers based on Tembusu virus NS5 and E gene were designed and a pair of primers of house-keeping gene β-actin was chosen. Then these amplified fragments were cloned into pMD18-T. Using the plasmids NS5-pMD18-T, E-pMD18-T and β-actin-pMD18-T as standard products, a real-time quantitative reverse transcription-polymerase chain reaction ( RT-PCR) was performed to construct the standard curves of NS5, E and β-actin gene and detect the sensitivity, specificity and repeatability. 【Result】 The results showed a precise linear relationship with a correlation coefficient of R²＞0.99. The detection limits was 10 copies of DNA plasmid reaction. The amplification curve showing a single peak could only been detected for Tembusu virus. The variation coefficient was less than 0.5% by within and between the group of repeatability tests. The clinical samples were detected 3 times by this method, and all results were positive.【Conclusion】 The developed real-time PCR assay was highly specific, sensitive, and reproducible and could be an available tool for diagnosis and monitoring of Tembusu virus in duck farms.

Key words:

Tembusu virus, NS5 gene, E gene, SYBR GreenⅠ real-time quantitative PCR assay, relative quantitative

于春梅, 刁有祥, 唐熠, 崔京腾, 高绪慧, 张颖, 鞠小军, 武利利. 坦布苏病毒荧光定量RT-PCR方法的建立[J]. 中国农业科学, 2012, 45(21): 4492-4500.

YU Chun-Mei, DIAO You-Xiang, TANG Yi, CUI Jing-Teng, GAO Xu-Hui, ZHANG Ying, JU Xiao-Jun, WU Li-Li. Fluorescence Quantitative RT-PCR Assay for Detection of Tembusu Virus[J]. Scientia Agricultura Sinica, 2012, 45(21): 4492-4500.

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