免标准曲线定量PCR技术的探讨  

Study on method of quantitative PCR without using standard curve

作　　者：克丙申[1] 陈英剑[1] 徐军[1] 齐法莲[1] 杜秀敏[1] 卢兆莲[1] 

机构地区：[1]济南军区总医院免疫科,山东济南250031

出　　处：《实用医药杂志》2007年第5期592-596,共5页Practical Journal of Medicine & Pharmacy

摘　　要：目的实际应用发现,传统的定量PCR方法误差较大不能满足科学研究以及临床精确定量的需要,本文拟探讨如何选择分析使用的技术资料以降低分析误差以及分析判断误差的主要来源。方法使用PCR动力学模型模拟实时定量PCR仪器跟踪测定资料,使用不同扩增时期的荧光数值资料计算起始模板数、分析误差,并根据仪器的荧光信号测定误差随机对资料加入误差后,分析结果的误差与研究误差的分布。结果使用指数扩增期内后期的5个资料点分析,可以获得当前仪器误差水平下,相对误差最小的结果。使用的资料点一旦包含有一个线性扩增期资料,结果的误差即显著增加。该文亦给出了一般性最后一次指数扩增循环的判定方法。结论免标准曲线的定量PCR方法将成为今后定量PCR的主要技术手段。Objective It has been found that traditional Q- PCR, which had a big error due to the problems from data processing and fluorescence signal detecting, couldn＇t satisfy the need of accurate quantification in scientific research, this article is to probe selection, analysis and application of technological data for improving accuracy of quantification, and to analysed and judge main source of error. Methods Quantification using quantitative PCR without standard curve is accepted but it cannot explain the dynamic model N =N0x（1 ＋p）c. During exponential amplification, using the model could accurately calculate the amplification efficiency and the initiative templates. But the data after exponential amplification badly deviated the dynamic model. Results Simulating AB1 7700, the error of initiative templates under 10% was gained when using datum of three effective digit fluorescence signals. It also was identified by the data of LightCycler. Efficiency of every sample can be calculated by using this method, which avoided supposing that all reactions have same amplification efficiency when using standard curve. Using this method to analyze PCR, the veracity depended on the precision of cycler to low fluorescence signal and the data coming from exponential phase. It was suggested that if the error of quantitative PCR results was controlled fewer than 10%, the measure veracity of cycler to low fluorescence signal wound attain to 3%.Conclusion The method of quantitative PCR without using standard curve could become a main techology tool of quantitative PCR.

关 键 词：荧光定量PCR 实时定量PCR PCR动力学模型 免标准曲线定量 

分 类 号：R446.61[医药卫生—诊断学]