SYBR Green Ⅰ Real-time PCR检测IFN-CSP对HepG2.2.15细胞内HBV-DNA影响

摘要

Objective To establish the SYBR Green Ⅰ Real-time PCR method for detecting the effect of IFN-CSP on HBV-DNA in HepG2.2.15 cells.Methods HBV-DNA was amplified and purified after extracted from HepG2.2.15 cell.Gradient dilution of PCR purified product was used as standard.Standard curve was established by SYBR Green Ⅰ Real-time PCR.The specificity,sensitivity,reproducibility and stability of SYBR Green Ⅰ Real-time PCR were analyzed.Effect of IFN-CSP on HBV-DNA in HepG2.2.15 cells was analyzed by SYBR Green Ⅰ Real-time PCR and the results were compared with Taqman Real-time PCR.Results The specificity,repeatability and stability of the SYBR Green Ⅰ Real-time PCR method were excellent.The linear range of the method was 107-102 copies,and the sensitivity of the method was 102 copies.IFN-CSP exhibited an inhibitory effect on HBV-DNA in HepG2.2.15 cells in a dose-dependent manner.There was no significant difference between the results of SYBR Green Ⅰ Real-time PCR and Taqman Real-time PCR.%目的 建立SYBR Green Ⅰ Real-time PCR检测HBV-DNA方法,并检测IFN-CSP对HepG2.2.15细胞内HBV-DNA影响.方法 提取HepG2.2.15细胞DNA,PCR扩增后纯化,PCR纯化产物梯度稀释作为标准品,采用SYBR Green Ⅰ Real-time PCR 检测,建立标准曲线,并分析方法的特异性、灵敏度、重复性和稳定性.运用建立的SYBR Green Ⅰ 方法检测IFN-CSP对HepG2.2.15细胞内HBV-DNA影响,并与Taqman方法进行比较.结果 SYBR Green Ⅰ Real-time PCR方法特异性、重复性及稳定性均较好,线性范围为107~102copies,检测灵敏度可达102copies.IFN-CSP对HepG2.2.15细胞内HBV-DNA具有抑制效果,且呈剂量依赖性.SYBR Green Ⅰ 方法与Taqman商业试剂盒检测结果差异无统计学意义.结论 SYBR Green Ⅰ Real-time PCR方法简便快速、结果准确、价格低廉,可以满足HBV-DNA拷贝数检测的需要.