细胞人基因组DNA对荧光实时定量PCR检测细胞培养液中HBV DNA的影响

Effect of human cellular genomic DNA on the HBV DNA in the culture medium detected by real-time fluorescence quantitative PCR

目的 研究细胞人基因组DNA 对荧光实时定量PCR(FQ-PCR)检测细胞培养上清液中HBV DNA 含量的可能干扰影响,提高细胞培养上清液中HBV DNA 定量检测的准确性.方法 取HBV DNA 阳性血清,以DMEM 培养基分别配制出HBV DNA 拷贝数为5×107/ml(高拷贝组)、5×105/ml(中拷贝组)、5×103/ml(低拷贝组)的不同样本组;各组内再分5 个亚组,分别加入终浓度为0(对照组)、12.5、25、50、100 μg/ml 的细胞人基因组DNA(提取自人肝癌细胞系HepG2).以不含HBV DNA 阳性血清的DMEM 培养基加入上述浓度细胞人基因组DNA 为空白组.采用基于TaqMan 技术的FQ-PCR 检测各组样本的HBV DNA 拷贝数并绘制HBV DNA 定量曲线.每组均重复检测10 次.根据HBV DNA 定量检测结果确定受干扰样本和未受干扰样本,分别计算其定量曲线线性期斜率和平均扩增效率.结果 高拷贝组中加入不同浓度细胞人基因组DNA 的各个亚组与相应对照组比较,HBV DNA 拷贝数差异均无统计学意义.中拷贝组中加入细胞人基因组DNA 浓度为50和100 μg/ml 的2个亚组,其HBV DNA 拷贝数结果明显高于相应对照组,增高可达50～100倍,且重复性差.低拷贝组中只有加入细胞人基因组DNA 浓度为12.5μg/ml的亚组可通过提高基线值取得与相应对照组接近的定量检测结果,而其他亚组HBV DNA 定量曲线指数扩增期斜率明显异常,无法确定其定量检测结果.空白组各亚组均未观察到明显的HBV DNA 指数扩增期.受干扰样本线性期斜率(-1.01±0.06)和平均扩增效率(90.0%±2.1%)与对照组样本(分别为-1.52±0.06,97.0%±0.4%)比较,差异均有统计学意义(P＜0.01).结论 细胞人基因组DNA 对应用FQ-PCR 技术进行的HBV DNA 定量检测可产生非特异性干扰,尤其在HBVDNA 拷贝水平较低时.在细胞培养上清液作HBV DNA 定量检测时应尽量去除细胞人基因组DNA.

Objective To investigate the interference effect of human cellular genomic DNA on the HBV DNA in the culture medium detected by real-time fluorescence quantitative PCR (FQ-PCR). Methods HBV DNA positive serum was diluted with DMEM culture medium to produce three test groups with HBV DNA levels of 5 × 107 copies/ml (high level group), 5 × 105 copies/ml (medium level group), and 5 × 103 copies/ml (low level group). Human cellular genomic DNA extracted from HepG2 cells was then added to the groups at final concentrations of 0 (control group), 12.5, 25, 50, and 100 μg/ml respectively, and those at the same concentrations added into the DMEM culture medium without HBV DNA were used as the blank groups. FQ-PCR based on TaqMan technique was used to detect the amount of HBV DNA copies and to establish quantitative curve of HBV DNA. The test was repeated for ten times in each group. According to the quantitative data of HBV DNA, the interfered and non-interfered samples were determined, and their amplification efficiency and the slope of linear phase of the quantitative curve were calculated respectively. Results In the high level group, no significant difference was detected in the amount of HBV DNA copies between the control and the subgroups added with different concentrations of human cellular genomic DNA. In the medium level group, the amount of HBV DNA copies in the 50 and 100 μg/ml subgroups was significantly higher than that in the control (increased 50- to 100-fold) with poor repeatability. In the low level group, the result could reach the level of the control by increasing the baseline value in the 12.5 μg/ml subgroup; while in the other subgroups, abnormal slope of linear phase of the quantitative curve could be observed, and the amount of HBV DNA copies was undetectable. No marked amplification of HBV DNA was detected in the blank groups. The slope of linear phase and amplification efficiency in the interfered samples (-1.01 ± 0.06 and 90.0% ± 2.1%) were significantly lower than those in the control (-1.52 ± 0.06 and 97.0% ± 0.4%; P < 0.01). Conclusions The human cellular genomic DNA unspecifically interferes with the detection of HBV DNA by FQ-PCR, particularly for the samples with a low level of HBV DNA copies. Removal of the human cellular genomic DNA from culture media prior to FQ-PCR is necessary for reliable HBV DNA quantitative detection.