凝胶成像系统对核酸扩增产物的定量检测

目的 建立一种PCR-凝胶成像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．将梯度标准血清(10^1～10^6拷贝／u1)分别进行40，35及32次循环次数的扩增，在琼脂糖电泳，EB染色后，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／ul，线性良好，而在10^4，10^5及10^6拷贝／ul三个梯度时直线上升缓慢，趋于平坦；32次循环时，10拷贝／ul未能检出，10^2～10^6拷贝／ul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UVIband的光密度定量分析指标中，以体积或体积百分比作为定量指标较好；本法除可用于常规PCR产物定量(HBV-DNA，TB—DNA)，也适用于RT-PCR(HCV-RNA)。

QUANTITATIVE DETECTION OF Gel DOCUMENTATION SYSTEMS ON NUCLEIC ACID AMPLIFICATION PRODUCTS

Objective: To establish polymerase chain reaction -gel documentation system (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 sera (101 - 10s copies/ul) wer 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 101-104 copies/ul, but line was nearly flat in 104 -106 copies/ul; when cycles were 32, 10 copies/ul-can't be detected, and linearity was good in 102 -106 copies/ul (r=0. 98). Conclusion:32 cycles were suitable cycle number for PCR-GDS; Among quantitative analysis of GDS, volume and volume percentage were better than others; This technology can not only be used as quantity for routine PCR, but only RT-PCR..