| 摘要 |
The invention provides an analytical method to reduce the quantitative analysis systematic error of real-time fluorescence thermal cycler for polymerase chain reaction (PCR) and the application thereof. In the method, t<SUB>n</SUB>, the amplifying time by which fluorescence intensity (R<SUB>n</SUB>) gets to a certain threshold, is set as the measuring index and initial template copy number (X<SUB>0</SUB>) is calculated according to the linear quantitative relation between t<SUB>n </SUB>and X<SUB>0</SUB>, shown as the formula 1: lnX<SUB>0</SUB>=-ln(1+E)(t<SUB>n</SUB>/t<SUB>c</SUB>)+lnK. Particularly, the method firstly needs to measure the values of t<SUB>n </SUB>of the standard samples whose X<SUB>0 </SUB>are known and protract the standard curve about lnX<SUB>0</SUB>~t<SUB>n</SUB>; then to measure the values of t<SUB>n </SUB>of samples and calculate the initial copy number of samples in terms of the standard curve. In the process of measuring t<SUB>n</SUB>, real-time fluorescence thermal cycler collects fluorescent signals in the pattern of continuous scanning and measures real-time fluorescence intensity. The instrument is set to measure fluorescence intensity (R<SUB>n</SUB>) in PCR tube in the frequency of any interval ranging from 0.01 second to 10 second in extending period. The dynamic curve about R<SUB>n</SUB>~t is automatically shown on the screen. On the dynamic curve, the amplifying time by which the intensity of fluorescent signals gets to the threshold is defined as t<SUB>n</SUB>. The method reduces the serious systematic error that the previous analytical pattern has and can be widely used in such fields as gene expression, gene engineering, drug curative effect, pathogen detection and genetically modified component detection, etc.
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