实时荧光定量PCR检测对虾白斑综合症病毒方法的建立

Establish of Real-time Fluorescent Quantitative PCR Assay for Detecting White Spot Syndrome Virus

[目的]根据对虾白斑综合症病毒（White Spot Syndrome Virus,WSSV）的保守序列,利用Primer5.0软件设计引物,建立了WSSV的荧光实时定量PCR检测体系。[方法]构建含有WSSV目扩增片段的质粒为标准品,进行定量PCR扩增,探索反应条件。[结果]确定了最佳退火温度（61℃）和最佳引物浓度（0.2μmol/L）,标准品浓度在8.8×1098.8×104个拷贝数之间有典型的＂s＂型扩增曲线,标准曲线中模板拷贝数（X）与Ct值的关系为Ct=-3.597lgX＋42.547,相关系数R2=0.993。[结论]该检测方法特异性强,对传染性皮下和造血器官坏死病毒（IHHNV）、肝胰腺细小病毒（HPV）没有扩增反应。

[Objective] A real-time fluorescent quantitative PCR assay utilizing SYBR green Ⅰdye was developed for detection and quantization of white spot syndrome virus particles isolated from infected shrimp.[Method]A pair of specific primers which could amplify a 221 bp fragment were designed based on Primer Express 5.0 software.The standards were established with plasmid containing the conserved region of WSSV.Reaction conditions were explored.[Result] The result showed that 61 ℃ was the best annealing temperature and 0.2 μmol/L was the best primers concentration.Typical ＂s＂ amplification curves were showed between concentrations of 8.8×109-8.8×104 copies.The linear relationship between virus concentration（X）and Ct was Ct=-3.597 lgX＋42.547 with the correlation coefficient R2 =0.993.[Conclusion]The primers were specific for WSSV and did not react with either Infectious Hypodermal and Hematopoietic Necrosis Virus（IHHNV）or Hepatopancreatic Parvovirus（HPV）.