摘要
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 develop a real-time qPCR assay for DNA copy of herpes simplex virus 2（HSV-2）. Methods Specific primers and Taqman fluorescent probes targeting the glycoprotein D gene fragment of HSV-2 were designed,based on which a real-time qPCR assay for DNA copy was developed using the genome of HSV-2 as a template, and verified for reproducibility and sensitivity. Results The linear range of the developed real-time qPCR assay was 1 × 10^7~1 × 10^2 copies / ml, with a r^2 value of more than 0. 99, while the sensitivity was 1 × 10^2 copies / ml. The coefficients of variation in inter- and intra-assays of references at various dilutions were 1. 70% ~ 5. 35% and 1. 70% ~ 4. 8%respectively. Conclusion Compared to the routine real-time PCR assay using plasmid as a template, the developed method showed high sensitivity, which might be used for the optimization of clinical diagnosis of genital herpes.
出处
《中国生物制品学杂志》
CAS
CSCD
2016年第10期1082-1086,共5页
Chinese Journal of Biologicals
基金
重大新药创制专项疫苗大平台子课题(2013ZX09402302-224)
