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目的建立检测Ⅱ型单纯疱疹病毒(herpes simplex virus 2,HSV-2)DNA拷贝的real-time qPCR方法。方法设计靶向HSV-2 g D片段的特异性引物和探针,以HSV-2基因组为模板,建立检测HSV-2 DNA拷贝的real-time qPCR方法,并进行重复性和灵敏度验证。结果 HSV-2 DNA拷贝在1×10~7~1×10~2 copies/ml范围内,线性良好,r~2>99%,灵敏度为1×10~2 copies/ml;不同稀释度参考品检测结果的批间变异系数为1.70%~5.35%,批内变异系数为1.70%~4.8%。结论与常规的以质粒为模板的real-time qPCR方法相比,以HSV-2基因组为模板建立的realtime qPCR方法的灵敏度更高,可用于生殖器疱疹临床诊断的优化。
Objective To establish a real-time qPCR method for detecting the herpes simplex virus 2 (HSV-2) DNA copy. Methods Specific primers and probes targeting HSV-2 g D fragment were designed and used to establish a real-time qPCR method to detect HSV-2 DNA copy with HSV-2 genome as template. The repeatability and sensitivity were also validated. Results The linearity of HSV-2 DNA copy was 1 × 10 ~ 7 ~ 1 × 10 ~ 2 copies / ml with r ~ 2> 99% and the sensitivity was 1 × 10 ~ 2 copies / ml. The coefficient of variation of the test results was 1.70% ~ 5.35%, and the coefficient of variation within the batch was 1.70% ~ 4.8%. Conclusion Compared with the conventional plasmid-based real-time qPCR method, the realtime qPCR method based on the HSV-2 genome is more sensitive and can be used for the clinical diagnosis of genital herpes.