| 摘要: |
| 以真鲷虹彩病毒(Red-sea bream iridovirus，RSIV)主要衣壳蛋白(Major capsid protein，MCP)的基因保守片段为靶序列，利用Primer Express 3.0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=-3.184 1gX+40.270，相关系数R2=0.996 9。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。 |
| 关键词: 真鲷虹彩病毒 实时定量PCR 衣壳蛋白基因 SYBR GreenⅠ |
| DOI： |
| 分类号: |
| 基金项目:国家质量监督检验检疫总局科研项目(2007IK267)和质检总局公益性项目(10-71)共同资助 |
|
| Development and application of a real-time PCR assay for the detection of red-sea bream iridovirus |
|
|
| Abstract: |
| 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 220×102 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. |
| Key words: sea bream iridovirus Real-time quantitative PCR Major capsid protein gene SYBR GreenⅠ |
