Kit for rapidly extracting gram-negative bacterium genome DNA and extraction method

A Gram-negative bacteria and extraction method technology, applied in the field of molecular biology, can solve the problems of large differences in nucleic acid extraction efficiency, achieve the effect of improving nucleic acid extraction efficiency, simple steps, and improving nucleic acid extraction purity

Active Publication Date: 2021-02-23
甘肃省科学院传感技术研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the nucleic acid extraction efficiency of various existing magnetic bead extraction methods is quite different, so it is necessary to improve the nucleic acid extraction technology of the magnetic bead method.

Method used

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  • Kit for rapidly extracting gram-negative bacterium genome DNA and extraction method
  • Kit for rapidly extracting gram-negative bacterium genome DNA and extraction method
  • Kit for rapidly extracting gram-negative bacterium genome DNA and extraction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1, a kit for rapidly extracting the genomic DNA of gram-negative bacteria, including a variety of working solutions, the specific preparation of each working solution is as follows:

[0037] (1) Working solution A: CTAB with a mass concentration of 0.8%, 2.5mol / L NaCl, a mass concentration of 2% Triton-X-100, 80mmol / L Tris-HCl (pH7.0-8.0), 15mmol / L EDTA , the mass concentration is 1% PVP, and the solvent is sterile deionized water.

[0038] (2) Working solution B: 2.0mol / L NaCl, 4.0mol / L guanidine isothiocyanate, 0.45mg / mL ribonuclease A, 0.07mol / L sodium citrate, the solvent is sterile deionized water.

[0039] (3) Working solution C is an ethanol solution with a volume fraction of 70%.

[0040] (4) Working solution D: 15mmol / L Tris-HCl, pH 7.0-8.0; solvent is sterile deionized water, pH 8.0.

[0041] (5) Magnetic bead binding solution: 1.5mol / L NaCl, 1.2mol / L guanidine hydrochloride, absolute ethanol with a volume ratio of 20%, isopropanol with a volume ratio ...

Embodiment 2

[0042] Embodiment 2, a kit for rapidly extracting the genomic DNA of Gram-negative bacteria, including a variety of working solutions, the specific preparation of each working solution is as follows:

[0043] (1) Working solution A: CTAB with a mass concentration of 0.5%, 3.5mol / L NaCl, Triton-X-100 with a mass concentration of 1%, 50mmol / L Tris-HCl (pH7.0-8.0), 25mmol / L EDTA, the mass concentration is 1% PVP, and the solvent is sterile deionized water.

[0044] (2) Working solution B: 1.5mol / L NaCl, 3.0mol / L guanidine isothiocyanate, 0.40mg / mL ribonuclease A, 0.1mol / L sodium citrate, the solvent is sterile deionized water.

[0045] (3) Working solution C is an ethanol solution with a volume fraction of 60%.

[0046] (4) Working solution D: 15mmol / L Tris-HCl, pH 7.0-8.0; solvent is sterile deionized water, pH 8.0.

[0047] (5) Magnetic bead binding solution: 0.5mol / L NaCl, 1.8mol / L guanidine hydrochloride, 30% absolute ethanol by volume, 60% isopropanol by volume, 3% polyeth...

Embodiment 3

[0048] Example 3, a kit for rapidly extracting the genomic DNA of Gram-negative bacteria, including a variety of working solutions, the specific preparation of each working solution is as follows:

[0049] (1) Working solution A: CTAB with a mass concentration of 1.0%, 1.5mol / L NaCl, Triton-X-100 with a mass concentration of 1%, 120mmol / L Tris-HCl (pH7.0-8.0), 8mmol / L EDTA, the mass concentration is 0.5% PVP, and the solvent is sterile deionized water.

[0050] (2) Working solution B: 1.0mol / L NaCl, 4.5mol / L guanidine isothiocyanate, 0.3mg / mL ribonuclease A, 0.08mol / L sodium citrate, the solvent is sterile deionized water.

[0051] (3) Working solution C is an ethanol solution with a volume fraction of 75%.

[0052] (4) Working solution D: 10mmol / L Tris-HCl, the solvent is sterile deionized water, pH8.0.

[0053] (5) Magnetic bead binding solution: 1.0mol / L NaCl, 1.5mol / L guanidine hydrochloride, 20% absolute ethanol by volume, 45% isopropanol by volume, 10% polyethylene gly...

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Abstract

The invention discloses a kit for rapidly extracting gram-negative bacterium genome DNA. The kit comprises a working solution A, a working solution B, a magnetic bead binding solution, a working solution C and a working solution D. The extraction method using the kit is simple, and mainly comprises the following four steps: bacterial lysis, magnetic bead adsorption, washing and elution. Monodisperse and superparamagnetic silicon oxide nano magnetic beads are adopted, other required reagents are conventional non-toxic reagents, the cost is low, the experiment safety is improved, and an extracted gram-negative bacterium genome DNA fragment is good in integrity, high in yield, high in purity and suitable for downstream molecular biology experiments such as PCR detection, nucleic acid hybridization and DNA library construction.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, in particular to a kit and an extraction method for rapidly extracting the genome DNA of gram-negative bacteria. Background technique [0002] Food safety is a public health security issue that is highly concerned by the world and directly related to people's livelihood, and food-borne pathogenic bacteria are one of the main reasons for frequent food safety incidents. Timely and accurate detection of pathogenic bacteria in food, Strengthening the screening and detection of harmful germs and effectively preventing food contamination is the fundamental measure to control the contamination of pathogenic bacteria in food and the spread of various infectious diseases. [0003] DNA extraction technology is the first and most critical step in the biosensor detection technology of food-borne pathogenic bacteria. Whether to obtain high-purity DNA will directly affect the safety and success of do...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1013Y02A50/30
Inventor 马莉萍李云霞聂莹莹左显维冯治棋马生龙任文斌
Owner 甘肃省科学院传感技术研究所
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