检测痕量单纯疱疹病毒Ⅱ型荧光定量PCR方法的建立

Development of Taq Man Real- time Fluorescence Quantitative PCR Assay for Detection of Herpes Simplex Virus Type 2

目的 建立一种高敏感度的检测痕量单纯疱疹病毒Ⅱ型（HSV-2）DNA的实时荧光定量PCR方法，为针对HSV-2潜伏感染的治疗性疫苗和抗病毒药物疗效提供一个评价方法。方法依据HSV-2糖蛋白D（gD）基因序列，使用序列比对软件，选取其保守区序列设计引物及TaqMan探针并进行筛选；以gD基因重组质粒pcDNA3-HSV-2gD作为标准模板，对该检测方法的反应条件进行优化，提高其检测特异性、敏感度和重现性。结果该检测方法特异性好，检测的最低拷贝数可达到2个拷贝，线性范围为2×10^0～2×10^8拷贝／反应，是普通PCR敏感度的10^3倍。该检测方法组间和组内变异系数均低于5％，说明该检测方法具有良好稳定性。结论本研究建立的TaqMan实时荧光定量PCR方法用于检测痕量HSV-2DNA具有特异、敏感、定量和重现性好的优点。

Objective A sensitive TaqMan fluorescence quantitative PCR assay for detecting herpes simplex virus type 2 （ HSV - 2） was developed for evaluating the treatment effect of antivirus medicines and therapeutic vaccines aiming at latent HSV - 2 infection. Methods Based on the nucleotide sequences of HSV - 2, the primers and probes were designed from the glycoprotein D gene of HSV - 2. The standard curve was generated by purified pcDNA3 - HSV - 2 gD of the recombined plasmid of gD gene. With the pcDNA3 - HSV - 2 gD as a template,the important influential factors including primers, probe, annealing temperature and primer concentrations were optimized for the highest sensitivity. Results This TaqMan fluorescence quantitative PCR assay showed good specificity for detecting HSV -2. The method can detect 2 copies/reaction which was 1000 - fold more sensitive than conventional PCR, and the linearity range was 2 × 10 - 2 ×10^8 copies/reaction. The intra - and inter - assay coefficients of variation were both less than 5%. Conclusion The TaqMan real - time fluorescent quantitative PCR assay was a specific, sensitive and stable method which could used for the quantitative detection of HSV - 2.