[1]刘青涛,李银,赵冬敏,等.一步法SYBR Green 实时定量RT-PCR检测坦布苏病毒方法的建立[J].江苏农业学报,2015,(04):829-833.[doi:10.3969/j.issn.1000-4440.2015.04.019]
　LIU Qing-tao,LI Yin,ZHAO Dong-min,et al.Development of a one-step SYBR Green-based real-time RT-PCR assay for detection of Tembusu virus[J].,2015,(04):829-833.[doi:10.3969/j.issn.1000-4440.2015.04.019]
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一步法SYBR Green 实时定量RT-PCR检测坦布苏病毒方法的建立()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2015年04期
页码:
829-833
栏目:
畜牧兽医·水产养殖
出版日期:
2015-08-31

文章信息/Info

Title:
Development of a one-step SYBR Green-based real-time RT-PCR assay for detection of Tembusu virus
作者:
刘青涛; 李银; 赵冬敏; 刘宇卓; 黄欣梅; 杨婧; 韩凯凯; 安凤娇
（江苏省农业科学院兽医研究所/农业部动物疫病诊断与免疫重点开放实验室/国家兽用生物制品工程技术研究中心，江苏南京210014）
Author(s):
LIU Qing-tao; LI Yin; ZHAO Dong-min; LIU Yu-zhuo; HUANG Xin-mei; YANG Jing; HAN Kai-kai; AN Feng-jiao
（Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences/Key Laboratory of Animal Disease Diagnostic and Immunology， Ministry of Agriculture/National Center for Engineering Research of Veterinary Bio-products, Nanjing 210014, China）
关键词:
坦布苏病毒; 实时定量RT-PCR; NS2A基因
Keywords:
Tembusu virus; real-time RT-PCR; NS2A gene
分类号:
S855.3
DOI:
10.3969/j.issn.1000-4440.2015.04.019
文献标志码:
A
摘要:
为建立快速检测坦布苏病毒（Tembusu virus，TMUV）的一步法SYBR Green实时定量RT-PCR方法，本研究根据TMUV NS2A基因的保守序列，设计并合成了1对特异性引物，以体外转录的NS2A基因作为标准品，采用一步法实时定量RT-PCR方法检测TMUV。结果显示该方法标准曲线的决定系数为0.997，具有良好的线性关系；灵敏度为1 μl 10拷贝，是常规RT-PCR方法的100倍；并且与其他病毒无交叉反应，批内和批间变异系数分别为 0.11%～0.30%和0.88%～1.31%，具有良好的特异性和稳定性。该方法对于TMUV人工感染鸭泄殖腔棉拭子的检测阳性率为100%，而常规RT-PCR检测的阳性率只有71%。因此，本研究所建立的一步法实时定量RT-PCR方法灵敏度高、重复性、特异性好，可以用于TMUV的临床检测。
Abstract:
A one-step SYBR Green-based real-time RT-PCR assay was established for detection of the Tembusu virus (TMUV) that recently has caused infectious disease in ducks in China, by using NS2A gene-specific primers and in vitro transcribed viral RNA. The results showed that the Ct value was strongly correlated (R2=0.997) with the logarithm of the transcribed viral RNA concentration, suggestive of a good linear relationship. The RT-PCR assay established was specific to TMUV, with no cross-reaction with other viruses. The inter- and intra-assay coefficients of variation ranged from 0.11% to 0.30% and 0.88% to 1.31%, respectively, indicative of good repeatability. The assay was 100 time more sensitive than the conventional RT-PCR, with a RNA detection-limit of 10 copies. All the cloacal swabs from TMUV infected ducks were detected TMUV positive by this real-time RT-PCR assay while the figure was only 71% by conventional RT-PCR. Therefore, the one-step SYBR Green-based real-time RT-PCR assay developed here provides a rapid means and effective way identify TMUV infection in ducks.

参考文献/References:


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备注/Memo

备注/Memo:
收稿日期：2014-12-16 基金项目：国家自然科学基金项目（31172345）；江苏省农业科技自主创新基金项目[CX（14）2091] 作者简介：刘青涛(1981-), 男，山东滨州人，博士，助理研究员，主要从事分子病毒学与免疫学研究。（Tel）025-84390047；（E-mail）taoqingliu2013@163.com 通讯作者：李银，（Tel）025-84391687；（Email）muziyin08@163.com
更新日期/Last Update: 2015-08-31