摘要
目的建立一种聚合酶链反应 (PCR) -凝胶图像处理系统定量分析核酸扩增产物的方法。方法 1.用PCR荧光定量分析仪实时监测 PCR扩增曲线 ,并选择合适的扩增循环次数 ;2 .将梯度标准乙型肝炎病毒 (HBV )阳性血清 (10 1 ～ 10 6 拷贝 /μl)分别进行 40 ,35及 32次循环次数的扩增 ,经琼脂糖电泳进行凝胶分析 ;3.选择凝胶分析软件中能客观反映核酸实际含量的最适参数 ,制定标准曲线。结果 40及 35次循环时 ,在 10 1 ～ 10 4拷贝 /μl线性良好 ,而在 10 4,10 5 及 10 6 拷贝 /μl 3个梯度时直线上升缓慢 ,趋于平坦。 32次循环时 ,10拷贝 /μl未能检出 ,10 2～ 10 6拷贝 / μl的 5个梯度的模板 (对数值 )与产物 (体积 )有良好的线性关系 (r=0 .97)。结论 32次循环线性范围较广 ,为最适循环次数 ;在 U VI band的吸光度定量分析指标中 。
Objective To establish polymerase chain reaction gel documentation systems (PCR GDS) technology for detecting nucleic acid amplification products. Methods 1.PCR amplification curve was observed with fluorescence quantitative PCR (FQ PCR), and suitable cycle numbers were selected. 2.Standard HBV DNA sera (10 1 10 6 copies/μl) were amplified 40, 35 and 30 cycles separately. The PCR products were gel electrophoried and EB stained, then analyzed with GDS. Results When cycles were 40 and 35, linearity was good in standard sera 10 1 10 4 copies/μl, but line was nearly flat in 10 4 10 6 copies/μl; when cycles were 32, 10 copies/μl can not be detected, and linearity was good in 10 2 10 6 copies/μl (r=0 98). Conclusion 32 cycles were suitable cycle number for PCR GDS. Among quantitative analysis of GDS, parameters of volume and volume percentage were better than others.
出处
《上海医学检验杂志》
北大核心
2001年第3期144-146,共3页
Shanghai Journal of Medical Laboratory Sciences
