[1]黄琦雯,王 庆,王英英,等.基因II型草鱼呼肠孤病毒TaqMan荧光定量 PCR检测方法的建立与应用[J].中国预防兽医学报,2017,(10):804-809.[doi:10.3969/j.issn.1008-0425.201704035]
 HUANG Qi-wen,WANG Qing,WANG Ying-ying,et al.Establishment and application of a TaqMan real-time PCR assay for detection of grass carp reovirus genotype Ⅱ[J].Chinese journal of preventive veterinary medicine,2017,(10):804-809.[doi:10.3969/j.issn.1008-0425.201704035]
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基因II型草鱼呼肠孤病毒TaqMan荧光定量 PCR检测方法的建立与应用()
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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2017年10
页码:
804-809
栏目:
诊断技术
出版日期:
2017-11-15

文章信息/Info

Title:
Establishment and application of a TaqMan real-time PCR assay for detection of grass carp reovirus genotype Ⅱ
文章编号:
1008-0589(2017)10-0804-06
作者:
 

黄琦雯王 庆王英英李莹莹石存斌任 燕高彩霞吴杰兴郑树城曾伟伟*

 (中国水产科学研究院珠江水产研究所 农业部渔药创制重点实验室/广东省水生动物免疫重点实验室,广东 广州510380)
Author(s):
 

HUANG Qi-wen WANG Qing WANG Ying-ying LI Ying-ying SHI Cun-bin REN Yan GAO Cai-xia WU Jie-xing ZHENG Shu-cheng ZENG Wei-wei*

 (Key Laboratory of Fishery Drug Development, Key Laboratory of Aquatic Animal Immune Technology,
Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Ministry of Agriculture, Guangzhou 510380, China)
关键词:
草鱼呼肠孤病毒基因Ⅱ型TaqMan 荧光定量PCR检测方法
分类号:
S852.65
DOI:
10.3969/j.issn.1008-0425.201704035
文献标志码:
A
摘要:
基因Ⅱ型草鱼呼肠孤病毒(GCRV-Ⅱ)是当前引起草鱼出血病暴发与流行的主要病原,为建立快速检测GCRV-Ⅱ的方法,本研究根据GCRV-ⅡRdRp基因的保守区域序列设计引物及TaqMan探针,经反应参数优化,建立了检测GCRV-Ⅱ的荧光定量PCR方法。结果显示,该方法仅对GCRV-Ⅱ的靶基因序列进行扩增,与其它非靶目标核酸均无交叉反应;其最低检出量为3 拷贝/μL病毒核酸,比普通PCR的敏感度高100倍;组内和组间重复试验变异系数均小于1 %。采用该方法检测60份草鱼出血病疑似样品,GCRV-Ⅱ阳性检出率为75 %(45/60),与细胞分离结果一致。应用该方法分析了GCRV-Ⅱ在不同细胞和不同培养方式下病毒的增殖含量,结果显示GSB和PSF细胞增殖量最大,达106拷贝/μL~107拷贝/μL病毒核酸,转瓶接种比常规的细胞培养瓶和培养板接种其病毒含量低2个数量级。本研究建立的GCRV-ⅡTaqMan荧光定量PCR方法具有高效、特异、敏感、可重复性强的优点,适用于GCRV-Ⅱ的临床快速检测和病毒定量分析。
Abstract:
The predominant genotype currently circulating in China is grass carp reovirus II (GCRV-II), which is the main pathogen responsible for grass carp hemorrhage disease outbreaks. To establish the rapid and sensitive assay for GCRV-II detection, a TaqMan real-time PCR was developed with a pair of primers and probe designed according to the conserved sequence of the GCRV-ⅡRdRp gene. The assay was specific to detect GCRV-II, but had no amplification from the DNA of other pathogens. The sensitivity of the method for GCRV-Ⅱ was 3 copies/μL, which was 100 times sensitive than that of conventional PCR. The reproducibility tests in intra-assay and inter-assay indicated that the coefficients of variation were both less than 1%. 60 suspected samples of grass carp hemorrhagic disease were detected, of which 45 were positive for GCRV-II(positive rate 75%), which were consistent with the results of cell separation. GSB and PSF cells were more suitable for proliferation of GCRV-II, and the virus titers could reach 106 copies/μL-107 copies/μL. The virus titers of inoculated with rollerbottle was about 100 times lower

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(本文编辑:李 爽)

更新日期/Last Update: 2017-11-06