摘要
在GenBank中搜寻虹彩病毒蛙病毒属主衣壳蛋白(MCP)基因序列并进行多重比较后,利用其中的保守序列,设计引物和Taqman探针,建立了虹彩病毒蛙病毒属实时荧光PCR检测方法。对荧光PCR反应条件进行优化,评估该检测方法的特异性、稳定性和重复性,利用10倍系列稀释法检验其灵敏度并与常规PCR做了比较。结果显示,该方法对虹彩病毒蛙病毒属成员(新加坡石斑鱼虹彩病毒除外)的检测有高度的特异性,与淋巴囊肿病毒、鳜鱼传染性脾肾坏死病毒、真鲷虹彩病毒、锦鲤疱疹病毒等其他水生动物病毒之间均无交叉反应,有良好的特异性,检测总DNA灵敏度为4.5×10-3pg/μL,比传统PCR的敏感度高出100倍,可对低病毒含量的样品进行准确检测。制作的标准曲线有极好的线性关系,且线性范围宽,相关系数为0.9984。组内和组间重复试验的Ct值标准偏差分别为1.2%和1.6%,有良好的重复性。与常规的PCR相比较,该方法具有快速、特异、敏感、可定量、可同时检测大量样品等优点,可用于出入境检疫和动物防疫监督部门对虹彩病毒蛙病毒属的疫情监测。
In order to establish a real-time PCR method for rapid detection of Ranavirus （family Iridoviridae）,the MCP gene of Ranavirus were down-loaded from GenBank. The gene sequences of MCP were aligned by using the biologic software and the specific primers and probe were designed in the conserved region. The primers, probe and the reactive condition were optimized to improve the sensitivity. The specificity, sensitivity,reproducibility of the method were estimated to establish a relatively quantitative standard curve. It was found that the specificity of this assay was high for member of Ranavirus （except for SGIV） without any cross-reactions with LCDV, RSIV, ISKNV, KHN and other commonly encountered viruses from aquatic animals. A good linear correlation was demonstrated in the standard curve for the real-time PCR assay. A minimum of 4. 5 ）〈 10^-3 pg/μL total DNA could be detected, which indicated a good sensitivity of the assay. The coefficients of variance （CV） were 1.2% and 1.6% for the intra-assay and inter-assay tests respectively,which indicated a good reliability. The results showed that this assay is a valuable complementary tool to the routine detection of Ranavirus. It has potential to apply in entry-exit inspection and quarantine.
出处
《华中农业大学学报》
CAS
CSCD
北大核心
2008年第2期172-176,共5页
Journal of Huazhong Agricultural University
基金
国家高技术研究发展计划(“863”计划)(2006AA100306)
国家质量监督检验检疫总局科研项目(2006IK003)
深圳出入境检验检疫局科研课题(SZK33-2005)资助
