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根据对虾白斑综合症病毒(White Spot Syndrome Virus,WSSV)的保守序列,利用Primer Express2.0软件设计引物和Taqman探针,建立了WSSV的实时定量PCR检测体系。构建含有WSSV目的扩增片段的质粒为标准品,进行定量PCR扩增,结果表明标准品浓度在1.5×10~7～1.5×10~1个拷贝之间有“S”型扩增曲线,检测限为15个拷贝。标准曲线中模板浓度(X)与Ct值的关系为:Ct=-3.77 lgX+43.73,相关系数R~2=0.9967。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)、斑节对虾杆状病毒(MBV)、肝胰腺细小病毒(HPV)以及对虾基因组DNA没有扩增反应。运用该方法对100尾对虾样品进行检测,14个对虾样品为阳性,而运用巢式PCR检测只有4个对虾样品为阳性。实时定量PCR方法检测WSSV,快速、特异、灵敏,在虾病的临床检测上具有重要意义。
According to the conserved sequences of White Spot Syndrome Virus (WSSV), Primer Express2.0 software was used to design primers and Taqman probes to establish real-time quantitative PCR detection system of WSSV. The plasmid containing WSSV amplified fragment was used as a standard sample and quantitative PCR was carried out. The results showed that there was a “S” amplification curve between 1.5 × 10 ~ 7 ~ 1.5 × 10 ~ 1 copies , The detection limit is 15 copies. The standard curve of the template concentration (X) and Ct values for the relationship: Ct = -3.77 lgX +43.73, correlation coefficient R ~ 2 = 0.9967. This assay is highly specific and does not amplify infectious subcutaneous and hematopoietic necrosis virus (IHHNV), M. bovis baculovirus (MBV), hepatophan parvovirus (HPV) and shrimp genomic DNA. Using this method, 100 shrimp samples were tested, and 14 shrimp samples were positive. Only 4 shrimp samples were positive by nested PCR. The detection of WSSV by real-time quantitative PCR is rapid, specific and sensitive, which is of great significance in the clinical detection of shrimp disease.