套式PCR检测体系扩增效率的分析及对假阳性的控制

Analysis of amplification efficiency of PCR detection system and control of false positive contamination

用扩增效率等量化的指标来控制PCR检测过程中出现的假阳性 ,调查了受假阳性污染的试剂在特定的PCR检测系统和环境中产生假阳性的PCR循环数界限 ,采用模板量与扩增倍数的线性回归关系分析系统的扩增效率并以此测定计算了两个不同循环数系统 (C2 7+15) 和C2 7+2 0 )的检测界限分别为 0 78fg和 0 0 6 5fg ,提出以此检测界限的 1/2量作为假阳性耐受量 ,两个不同循环数系统的假阳性耐受量分别为 5 80个和 48个拷贝。

The experiment was to control appearance of false positive phenomenon in a nested PCR detection system by quantity indexes.The limit of cycle numbers,over which the false positive phenomenon of PCR could occur ,was firstly investigated for the given PCR detection system in the experimental environment.The amplification effciency of PCR was analyzed by regression relationship between the templates quantity and the amplification multiples of PCR,so that the detection limits of the nested PCR systems with deferent cycle numbers(C 27+15 and C 27+20 ) were determined,0.78 fg and 0.065 fg,respectively.A conception or index,false positive contamination endurance quality, which was a half of the PCR detection limit was recommended to use in detection by PCR and in this experient the indexes were 0.39 fg(C 27+15 ) and 0.033 fg(C 27+20 ) respectively.