DNA extraction-free quantitative PCR method for detection of plasma free DNA level by using Alu homologous gene B1 gene
A homologous gene and gene technology, which is applied in the field of quantitative PCR for detecting plasma cell-free DNA using the homologous gene B1 gene of Alu gene without DNA extraction, can solve the problems of lag, cumbersome detection, lack of specificity, etc.
Inactive Publication Date: 2016-04-06
SHANGHAI UNIV OF SPORT
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[0005] Considering the prevalence and harm of overtraining, and the currently used monitoring indicators not only have many indicators, but also are cumbersome, time-consuming, lack of specificity, and often lag behind the reality, combined with the research results in recent years, the introduction of plasma cell-free DNA Level is an indicator to reflect overtraining
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[0021] Quantitative PCR detection method of plasma cell-free DNA levels reflecting overtraining in rats and mice.
[0022] Take blood before training and immediately after training, choose 20ml of venous blood, anticoagulant with heparin or EDTA. Centrifuge and collect plasma. The DNA used as the standard and the 5-fold diluted plasma of each sample were added to the reaction system of quantitative PCR. MaximaSYBRGreen / ROXqPCR MasterMix (2x), 12.5ml; forward and reverse primers (50mM), 0.2ml each; standard or 5-fold diluted sample, 1.0ml each; DNase-free water, 11.1ml. The free DNA level of the sample was calculated according to the concentration of the standard.
[0023] Comparing the plasma free DNA levels before training and immediately after training, if the increase of several times to tens of times strongly indicates the possibility of overtraining in mice, the more significant the increase, the more likely it is the overtrainer and the degree of overtraining is more s...
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The invention provides a quantitative PCR method for detection of plasma free DNA level reflecting the overtraining of rats and mice mouse. The amplification primers are particularly selected from homologous gene B1 repetitive sequence gene with the richest expression of Alu in human genes; and the method has higher sensitivity and does not need DNA extraction. The method is as below: immediately sampling blood before and after training, selecting 20ml of venous blood, heparin or EDTA anticoagulant; centrifuging and collecting plasma; adding a DNA standard sample and 5 times diluted plasma samples into a quantitative PCR reaction system: 12.5ml of MaximaSYBRGreen / ROXqPCRMasterMix (2x), 0.2ml of a forward primer and 0.2ml of a reverse primer (50mM), 1.0ml of a standard sample or 1.0ml of 5 times diluted samples, and 11.1ml of DNA enzyme-free water; calculating the free DNA level according to the concentration of the standard sample; and comparing the immediate plasma free DNA levels before and after training, wherein several times to tens of times strong increase suggests the possibility of overtraining of rats and mice. The quantitative PCR method can be used as a detection method of plasma free DNA level, and can reflect the overtraining situation of rats and mice.
Description
technical field [0001] The present invention aims to provide a quantitative PCR detection method for plasma free DNA levels that can reflect the overtraining situation of mice and mice. Plasma cell-free DNA levels can be detected. If you want more accurate values and dynamic response to the overtraining process, you can choose to collect venous blood before training, immediately after training, and at different times after training, detect the level of plasma free DNA, and dynamically observe and compare. Background technique [0002] The current methods for detecting the level of plasma free DNA include comet detection or single-cell gel electrophoresis test, fluorescence quantitative determination of plasma DNA and quantitative PCR method. The advantage of the first method is that it can detect DNA damage at the single-cell level, but the cells obtained may not reflect the total DNA damage in plasma; the second method can only be carried out after plasma DNA extraction,...
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Inventor 王晓慧
Owner SHANGHAI UNIV OF SPORT