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当前位置: 首页 > 文章 > 真鲷虹彩病毒实时定量PCR检测方法的建立与应用 渔业科学进展 2011,32 (2) 111-116
Position: Home > Articles > Development and application of a real-time PCR assay for the detection of red-sea bream iridovirus Progress in Fishery Sciences 2011,32 (2) 111-116

真鲷虹彩病毒实时定量PCR检测方法的建立与应用

作  者：
赵玉然;谭乐义;刘荭;赵巍;梁成珠;史秀杰;徐彪;朱来华;何俊强;岳志芹
单  位：
山东出入境检验检疫局技术中心;深圳出入境检验检疫局
关键词：
真鲷虹彩病毒;实时定量PCR;衣壳蛋白基因;SYBR GreenⅠ
摘  要：
以真鲷虹彩病毒(Red-sea breamiridovirus,RSIV)主要衣壳蛋白(Major capsid protein,MCP)的基因保守片段为靶序列,利用Pri mer Express3.0软件设计定量PCR引物,建立了RSIV的SYBR GreenⅠ实时定量PCR检测方法。将RSIV MCP基因连接pMD18-T载体,构建重组质粒,经过梯度稀释后作为标准品,根据标准品拷贝数(X)与Ct值的关系绘制了标准曲线,为Ct=-3.1841gX+40.270,相关系数R2=0.9969。熔解曲线分析表明,定量PCR产物的Tm值为82.5℃。该方法的检测限为2.20×102拷贝/反应,对流行性造血器官坏死病毒、淋巴囊肿病毒、蛙病毒3、甲鱼虹彩病毒都没有扩增反应,具有特异性。利用该方法对84批海水鱼类(石鲽、大菱鲆、鲈鱼)进行检测,其中5批鱼样品感染RSIV,并利用标准曲线对病毒含量进行了定量分析。
译  名：
Development and application of a real-time PCR assay for the detection of red-sea bream iridovirus
作  者：
ZHAO Yu-ran1 TAN Le-yi1 LIU Hong2 ZHAO Wei1 LIANG Cheng-zhu1SHI Xiu-jie2 XU Biao1 ZHU Lai-hua1 HE Jun-qiang2 YUE Zhi-qin1(1Shandong Technical Center of Inspection and Quarantine Bureau,Qingdao 266002)(2Key Laboratory of Aquatic Animal Diseases,Shenzhen Entry-Exit Inspection and Quarantine Bureau,518010)
关键词：
Red-sea bream iridovirus Real-time quantitative PCR Major capsid protein gene SYBR GreenⅠ
摘  要：
A sensitive and specific SYBR GreenⅠreal-time PCR assay for the detection of red-sea bream iridovirus(RSIV)was established.The real-time PCR primers were designed according to the conserved region of major capsid protein(MCP)gene by using Primer Express 3.0 software.The RSIV MCP gene was inserted into pMD18-T vector to construct the recombinant plasmid.The resulted plasmid was serially diluted and used as the standards.The relationship between plasmid copy number(X)and Ct value was described as a standard curve:Ct=-3.184 lgX+40.270(with an R2 value of 0.996 9).The detection limit of the assay was 2.20×102 virus copies per reaction.The assay showed specificity and could not be amplified with RSIV and epizootic haematopoietic necrosis virus(EHNV),lymphocystis disease virus(LCDV),frog virus 3(FV 3),or soft-shelled turtle iridovirus(STIV).The Tm of the specific product was obtained as 82.5 ℃ through the melting curve analysis.This assay was applied in detecting whether the sea fish samples(stone flounder,turbot,and weever)of 84 batches were infected by RSIV.It was found that the samples of 5 batches presented positive signals,and the virus was quantified by using the obtained standard curve.The results indicate that the real-time PCR assay is specific,sensitive,fast,and accurate and it has great potential in detecting RSIV and studying the pathogenic mechanism of RSIV.

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