中文摘要：

目的建立一种快捷、灵敏、特异的Ⅱ型单纯疱疹病毒（Herpes simplex virus 2,HSV-2）实时荧光定量PCR检测体系。方法设计HSV-2糖蛋白E基因特异性引物,PCR扩增gE基因片段,并与pGEM-T-easy载体连接,构建pGEM-T-easy-gE2重组质粒。以连续10倍稀释的浓度梯度制备PCR体系标准品质粒,应用SYBR Green法建立gE2real-time PCR方法并优化引物浓度、Mg2＋浓度、退火温度和循环程序等条件,绘制标准曲线,并验证方法的敏感性、特异性和稳定性;用该方法检测HSV-2感染小鼠生殖道拭子标本。结果 gE2real-time PCR的标准品线性关系良好（r2=0.997）,其他妇科常见病原体无特异扩增;方法的最低检测质粒浓度为3.85×10^1 copy/μl,试验的变异系数〈1%,检测30份小鼠HSV-2感染生殖道拭子标本的阳性率为96.7%,高于常规PCR方法（80.0%）。结论gE2real-time PCR法检测HSV-2具有灵敏、特异、重复性好,可用于HSV-2定性分析和病毒载量检测。

英文摘要：

Objective To develop a rapid,semiautomated,quantitative fluorescence-based PCR assay to detect herpes simplex virus 2DNA. Methods Specific primers for the glycoprotein E gene（gE2）of HSV-2were designed.gE2 was amplified with a polymerase chain reaction（PCR）and then ligated into a pGEM-T-easy vector to construct the recombinant plasmid pGEM-T-easy-gE2.After gE2 was identified and verified using PCR,10-fold serial dilutions of plasmid pGEM-T-easy-gE2 were used as standard templates for SYBR Green real-time PCR to generate a standard curve to quantify the number of copies of gE2.Reaction conditions,primer concentrations,the Mg2＋concentration,and annealing temperatures and cycles were optimized,resulting in an increase in the amount of the desired PCR product.The sensitivity,stability,and specificity of the assay were tested and HSV-2was detected in genital tract swabs from infected mice. Results A good linear correlation was evident in the standard curve for the real-time PCR assay,and R-squared was 0.997.The coefficient for the plotted standard curve was 0.997,indicating a linear relationship.The assay had a detection limit of 3.85×101 copies/μl,and its coefficient of variation was less than 1%in reproducible assays.The gE2real-time PCR assay was highly specific for HSV-2and did not cross-react with DNA templates from HSV-1,Trichomonas vaginalis,the EB virus,and CMV.The assay had a higher rate of detecting HSV-2in 30 genital tract swabs（96.7%）than conventional PCR did（80.0%）. Conclusion Results indicated that the gE2real-time PCR assay is highly specific,it is highly sensitivity,and it can be used to detect and quantitatively analyze HSV-2.