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DNA extraction-free quantitative PCR method for detection of plasma free DNA level by using Alu repetitive sequence gene amplimer

A repetitive sequence and gene amplification technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of less blood consumption, free DNA extraction and sensitivity, etc., and achieve exercise ability and performance improvement, avoiding the effect of exercise-induced immunosuppression

Inactive Publication Date: 2016-04-06
SHANGHAI UNIV OF SPORT
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Considering that the most advantageous quantitative PCR method among the various methods for detecting plasma free DNA levels also has the reality that the extraction of free DNA is not the best and the sensitivity is not the best, a method that does not require plasma DNA extraction is needed in application. A less bloody and sensitive test method proposed

Method used

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  • DNA extraction-free quantitative PCR method for detection of plasma free DNA level by using Alu repetitive sequence gene amplimer
  • DNA extraction-free quantitative PCR method for detection of plasma free DNA level by using Alu repetitive sequence gene amplimer
  • DNA extraction-free quantitative PCR method for detection of plasma free DNA level by using Alu repetitive sequence gene amplimer

Examples

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Embodiment 1

[0023] Quantitative PCR detection of human plasma cell-free DNA levels reflecting overtraining in athletes.

[0024] Blood was collected before and after training, 20 microliters of fingertip blood or venous blood was selected, and anticoagulated 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), 12.5 μl; forward and reverse primers (50 μmol), 0.2 μl each; standard or 5-fold diluted sample, 1.0 μl each; DNase-free water, 11.1 µl. The free DNA level of the sample was calculated according to the concentration of the standard.

[0025] Comparing the plasma cell-free DNA levels before training and immediately after training, if it increases several times to tens of times, it can be regarded as a possible candidate for overtraining.

Embodiment 2

[0027] The method is basically the same as in Example 1, except that the detection of plasma free DNA levels at multiple time points after training is added, and the plasma free DNA levels or the recovery process of overtraining are dynamically observed.

[0028] Considering the practical application value of the quantitative PCR detection method we established in athletes overtraining, we applied this method to wrestling and boxing athletes, and detected wrestlers immediately after a high-intensity training, the next day and A few days later, as well as the plasma free DNA levels of boxers before and after the high-intensity and heavy-load closed training in preparation for the competition, and the seven major indicators currently used to reflect over-training, such as blood testosterone levels, cortisol levels, and testosterone levels. Cortisol ratio, morning pulse, Hb level, blood urea, and serum creatine kinase were analyzed for correlation. It was confirmed that plasma fr...

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Abstract

The invention provides a quantitative PCR method for detection of plasma free DNA level reflecting the overtraining of athletes. The amplification primers are particularly selected from Alu repetitive sequence gene with the richest expression in human genes; and the method has higher sensitivity and does not no need DNA extraction. The method is as below: immediately sampling blood before and after training, selecting 20 mul of finger tip blood or 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: 12.5 mul of MaximaSYBRGreen / ROXqPCRMasterMix (2x), 0.2mul of a forward primer and 0.2mul of a reverse primer (50 muM), 1.0 mul of a standard sample or 1.0 mul 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 overtraining. The quantitative PCR method can be used as a detection method of plasma free DNA level, and can reflect the overtraining situation of athletes.

Description

technical field [0001] The present invention aims to provide a quantitative PCR detection method for the free DNA level in human plasma that can reflect the athlete's over-training situation. The plasma cell-free DNA level can be detected. If you want more accurate values ​​and dynamic response to the overtraining process, you can choose to take fingertip blood or venous blood before training, immediately after training, and at different times after training to 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 a...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 王晓慧
Owner SHANGHAI UNIV OF SPORT