Reagent and method for quick release of nucleic acid

A nucleic acid release and reagent technology, which is applied in the field of rapid nucleic acid release reagents, can solve the problems of high price and limited wide application, and achieve the effects of simple operation, avoiding differences and errors, and stable experimental results

Active Publication Date: 2011-10-19
SANSURE BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The liquid method to extract nucleic acid is basically discarded because it requires toxic organic solvents such as chloroform and phenol; the spin column method is currently the most widely used, and the extraction steps are simple compared to the liquid method; the magnetic bead method has only been developed in recent years The nucleic acid extraction method, its series of reagents do not contain toxic organic solvents such as chloroform, phenol, etc. Compared with the spin column method, the extraction steps are simpler, no centrifugation is required, and it is easy to automate, but the series of reagents used in production are basically foreign monopoly, the price is very expensive, which limits its wide application in our country

Method used

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  • Reagent and method for quick release of nucleic acid
  • Reagent and method for quick release of nucleic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1 is used for the fluorescent quantitative PCR detection embodiment of HBV DNA

[0019] Preparation of nucleic acid releasing reagent: 0.1 mM / L surfactant peptide was dissolved in 80 mM / L KCl sterile aqueous solution, 0.1% (mass volume ratio) SDS and 0.5% (volume) ethanol were added. Said percentage is based on the volume of sterile water (the same below).

[0020] Fluorescent quantitative PCR detection of HBV DNA: suck 5 microliters of the nucleic acid release reagent and 5 microliters of the sample to be tested (standard substance, negative, positive control, positive serum or plasma of HBV DNA of unknown concentration) with a suction nozzle with a filter core, Add it to the PCR reaction tube, pipette the tip of the pipette 5 times, mix well, and let it stand for about 5 minutes. Use a filter tip to suck up 40 microliters of the prepared PCR reaction solution, and use it in Stratagene Mx3000P or ABI7300 / 7500 series PCR. Real-time fluorescent quantitative P...

Embodiment 2

[0027] Embodiment 2 is used for the fluorescent quantitative PCR detection of HCV RNA

[0028] Nucleic acid release reagent preparation: 0.05mM / L surfactant peptide was dissolved in 100mM / L KCl sterile aqueous solution, and 0.01% (mass volume ratio) LLS and 0.05% (volume) ethanol were added.

[0029] Aspirate 5 microliters of the nucleic acid release reagent and 5 microliters of the sample to be tested (standard, negative and positive controls, serum or plasma known to be positive for HCV RNA) with a suction nozzle with a filter core, add them to the PCR reaction tube, and pipette tip Pipette 8 times, mix well, and let stand for about 5 minutes, then use a filter nozzle to draw 40 microliters of the prepared PCR reaction solution, and perform real-time fluorescent quantitative PCR amplification on a Stratagene Mx3000P or ABI7300 / 7500 series PCR instrument.

[0030] The HCV PCR reaction solution consists of primers, probes, dNTPs, 5×PCR buffer, sterilized purified water, ROX so...

Embodiment 3

[0036] Embodiment 3 is used for the fluorescence quantitative PCR detection of venereal disease serial pathogen DNA

[0037] Preparation of the nucleic acid releasing reagent: prepare the nucleic acid releasing reagent according to the ratio of Example 1, and dilute the nucleic acid releasing reagent with water at a ratio of 2:3 before use.

[0038] Fluorescent quantitative PCR detection of pathogenic DNA of venereal disease series: wipe the urethral opening with a cotton test piece, then insert another cotton test piece into the urethra and turn it gently, take it out, put the test piece in an EP tube containing 0.5ml of normal saline, stir and squeeze After drying, the swab was discarded, and 5 μl of the above-mentioned secretion-containing sample was sucked with a filter nozzle, added to the nucleic acid release reagent, pipetted 10 times with a pipette tip, mixed, and left to stand for about 5 minutes, then used Take out 40 μl of the prepared PCR reaction solution with a f...

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Abstract

The invention relates to a reagent and a method for the quick release of nucleic acid. The reagent for releasing the nucleic acid is prepared by dissolving 0.01 to 0.5 milliliter per liter (the concentration of the surface bioactive peptide in the sterile aqueous solution of KCl is 0.01 to 0.5 mm / l) of the surface bioactive peptide in 50 to 200 milliliter per liter of sterile aqueous solution of KCl, and adding 0.01 to 2 percent (the concentration of the SDS, the LLS or the Chelex-100 in sterile water is 0.01 to 2 percent) of the SDS, the LLS or the Chelex-100, and 0.05 to 1 percent (volume) of ethanol, wherein the percentages are calculated based on the volume of the sterile water. The method for releasing the nucleic acid comprises the following steps of: taking the reagent for releasing the nucleic acid, and performing equivalent dilution on the reagent by using the sterile water according to different experimental purposes; balancing the diluted reagent to the room temperature, and then mixing the reagent uniformly; sub-packaging the mixture into a sample tube to be tested; adding a sample to be treated in the sample tube; and absorbing and beating the sample repeatedly for 5 to 10 times by a pipettor. The reagent for releasing the nucleic acid is safe to use and has a low production cost; and the method for releasing the nucleic acid is simple and convenient to operate.

Description

technical field [0001] The present invention relates to a nucleic acid rapid release reagent and method. Background technique [0002] At present, nucleic acid extraction methods have mainly experienced three development stages: liquid method-spin column method-magnetic bead method. The liquid method to extract nucleic acid is basically discarded because it requires toxic organic solvents such as chloroform and phenol; the spin column method is currently the most widely used, and the extraction steps are simple compared to the liquid method; the magnetic bead method has only been developed in recent years The nucleic acid extraction method, its series of reagents do not contain toxic organic solvents such as chloroform, phenol, etc. Compared with the spin column method, the extraction steps are simpler, no centrifugation is required, and it is easy to automate, but the series of reagents used in production are basically foreign Monopoly, the price is very expensive, which l...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
Inventor 戴立忠
Owner SANSURE BIOTECH INC
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