重组溶瘤2型单纯疱疹病毒荧光定量PCR检测方法的建立与验证

Establishment and Verification of Fluorescence Quantitative PCR for Detection of Recombinant Oncolytic Herpes Simplex Virus Type 2

目的为了建立高效、敏感的针对重组溶瘤2型单纯疱疹病毒(oHSV2)的荧光定量PCR方法。方法依据oHSV2的基因序列,登录Beacon Designer 8.0软件设计引物,提取前期已重组好的阳性质粒pHG52d34.5CMVhGM-CSF,测定浓度并换算后梯度稀释为荧光定量PCR的模板,进行PCR反应。结果经过多次调整实验的反应条件和反应体系,熔解曲线为单一峰,该病毒与野生型Ⅰ型单纯疱疹病毒、野生型Ⅱ型单纯疱疹病毒等没有交叉反应。oHSV2最低检测限为1000 copies/μL,R^2可达0.997,批内及批间变异系数分别为0.357%~1.156%和1.06%~3.35%,体液(血液)中预加的oHSV2的回收率为96.6%~109.3%。结论成功建立了oHSV2的SYBR Green实时荧光定量PCR方法,可特异而灵敏地检测血浆中的oHSV2。

Objective To establish an efficient and sensitive fluorescence quantitative PCR method for the detection of recombinant oncolytic herpes simplex virus type 2(oHSV2). Methods Specific primer was designed according to the sequence of oHSV2 gene by using Beacon Designer software.The positive control plasmid DNA was extracted and diluted to the standard for fluorescence quantitative PCR after concentration determination and conversion.Results Melting curve was drawn by optimizing the reaction conditions and reaction system.The melting curve was a specific single peak,without primer dimer or specific amplification peak.In addition,no cross reaction with wild herpes simplex virus type I typeⅡ was detected.Its minimum detection limit was 1000 copies/μL,R^2 could be developed 0.997,and the coefficient of variation within and between batches was 0.357%~1.156% and 1.06%~3.35%.The recovery of the pre-loaded oHSV2 in plasma was 96.6%~109.3%.Conclusion The SYBR Green real-time fluorescence quantitative PCR method is successfully built,which can be used to detect oHSV2 in blood samples specifically and sensitively.