| 锦鲤疱疹病毒实时荧光定量PCR检测方法的建立及应用 |
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| 引用本文： | 范万红,刘荭,岳志芹,吕建强,何俊强,江育林.锦鲤疱疹病毒实时荧光定量PCR检测方法的建立及应用[J].中国海洋大学学报(自然科学版),2007,37(5):785-788,722. |
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| 作者姓名： | 范万红 刘荭 岳志芹 吕建强 何俊强 江育林 |
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| 作者单位： | 1. 深圳出入境检验检疫局,广东,深圳,518001
2. 山东出入境检验检疫局,山东,青岛,266002 |
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| 基金项目： | 国家质量监督检验检疫总局科研项目 |
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| 摘 要： | 研究建立并完善了Taqman实时荧光定量PCR检测锦鲤疱疹病毒（KHV）的方法。首先通过选取KHV病毒TK基因的保守序列（GenBank AB182940）,利用PRIMER EXPRESS 2.0设计引物和探针。其中Taqman探针的5’端标记FAM,3’端标记TAMRA。然后利用梯度稀释的阳性样品克隆质粒进行定量PCR反应,制作标准曲线,得到病毒拷贝数与Ct值的关系为：Ct=-3.45 lgX＋42.73（相关系数R2=0.989）。通过试验检测得到实时荧光定量PCR对KHV的灵敏度为32个病毒核酸拷贝数,同时,设计的引物和探针对于鱼类细胞系和其它鱼类病毒没有扩增反应,表现出较好的特异性。实验结果表明,实时荧光定量PCR检测KHV的方法,具有特异性好、灵敏度高的特点,可以缩短检测时间,提高检测速度,非常适合口岸进出口检验检疫的要求。
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| 关 键 词： | 锦鲤疱疹病毒 实时荧光定量PCR 检测 |
| 文章编号： | 1672-5174（2007）05-785-05 |
| 修稿时间： | 2006-11-062007-01-04 |
Establishment and Application of the Real-Time PCR Detection Assay for Koi Herpesvirus |
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| FAN Wan-Hong,LIU Hong,YUE Zhi-Qin,LU Jian-Qiang,HE Jun-Qiang,JIANG Yu-Lin.Establishment and Application of the Real-Time PCR Detection Assay for Koi Herpesvirus[J].Periodical of Ocean University of China,2007,37(5):785-788,722. |
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| Authors: | FAN Wan-Hong LIU Hong YUE Zhi-Qin LU Jian-Qiang HE Jun-Qiang JIANG Yu-Lin |
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| Abstract: | A real-time quantitative PCR assay was developed for detection of koi herpesvirus(KHV).The conservative fragment of thymidine kinase(TK)gene of KHV was selected and the primers and probes were designed using the software PRIMER EXPRESS 2.0.The Taqman probes were labelled with fluorescent dyes FAM at the 5' end and TAMRA at the 3' end.The standard curve was prepared based on the linear relationship between the amount of input plasmid DNA(X) and cycle threshold(Ct).For KHV virus,Ct=-3.45 lgX 42.73(correlation coefficent R2=0.989).The real-time assay had a detection limit of 32 gene copies for KHV virus.The primers and Taqman probe used in this assay were shown to be specific for KHV and did not amplify other fish DNA viruses.Twenty four fish samples collected were subjected to real-time PCR and the results showed that four were KHV-positive and the others were negative.The results demonstrate that the real-time PCR method can be used as an effective detection and quantification method for KHV and it is amenable to high-throughout assay for its specificity,sensitivity and rapidity. |
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| Keywords: | koi herpesvirus real-time quantitative PCR taqman probe |
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