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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: 2010-05-26
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] Example 1 Fluorescence quantitative PCR detection example for HBV DNA

[0019] Preparation of nucleic acid release reagent: 0.1 mM / L surface active peptide is dissolved in 80 mM / L KCl sterile aqueous solution, and 0.1% (mass to volume ratio) SDS and 0.5% (volume) ethanol are added. The percentage is calculated based on the volume of sterile water (the same below).

[0020] HBV DNA fluorescence quantitative PCR detection: suck 5 microliters of the nucleic acid release reagent and 5 microliters of the test sample (standard, negative, positive control, unknown concentration of HBV DNA-positive serum or plasma) with a filter tip, Add to the PCR reaction tube, pipette the tip 5 times, mix well, let stand for about 5 minutes, use the filter nozzle to suck 40 microliters of the prepared PCR reaction solution, in Stratagene Mx3000P or ABI7300 / 7500 series PCR Perform real-time fluorescence quantitative PCR amplification on the instrument.

[0021] The concentration and ratio of each ...

Embodiment 2

[0027] Example 2 Fluorescence quantitative PCR detection for HCV RNA

[0028] Preparation of nucleic acid release reagent: 0.05mM / L surface active peptide is dissolved in 100mM / L KCl sterile aqueous solution, and 0.01% (mass to volume ratio) LLS and 0.05% (volume) ethanol are added.

[0029] Use a suction nozzle with a filter element to suck up 5 microliters of the nucleic acid release reagent and 5 microliters of the sample to be tested (standard, negative and positive control, known HCV RNA positive serum or plasma), and add it to the PCR reaction tube with a pipette tip Pipette 8 times, mix well, and after standing for about 5 minutes, use a filter nozzle to suck 40 microliters of the prepared PCR reaction solution, and perform real-time fluorescence quantitative PCR amplification on the Stratagene Mx3000P or ABI7300 / 7500 series PCR instrument.

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

Embodiment 3

[0036] Example 3 Fluorescence quantitative PCR detection for DNA of pathogens of STD series

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

[0038] Fluorescence quantitative PCR detection of pathogen DNA of STD series: Wipe the urethral opening with a cotton test piece, then insert another cotton test piece into the urethra and gently rotate it and take it out. Place the test piece in an EP tube containing 0.5ml of saline and stir and squeeze it. After drying, discard the swab, use a suction nozzle with a filter to suck 5μl of the above-mentioned secretion-containing sample, add it to the nucleic acid release reagent, pipette 10 times with a pipette tip, mix well, and let it stand for about 5 minutes. The suction nozzle with filter element sucks 40μl of the prepared PCR reaction solution, and performs real-time flu...

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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 invention relates to a reagent and method for rapid release of nucleic acid. Background technique [0002] At present, nucleic acid extraction methods have mainly experienced three stages of development: liquid method-spin column method-magnetic bead method. The liquid method to extract nucleic acids 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 simpler than the liquid method; the magnetic bead method has only been developed in recent years Nucleic acid extraction method, its series of reagents do not contain chloroform, phenol and other toxic organic solvents. Compared with the spin column method, the extraction step is simpler, does not require centrifugation, and is easy to realize automation. However, the series of reagents used are basically foreign The monopoly is very expensive, which limits its wide applicati...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 戴立忠
Owner SANSURE BIOTECH INC
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