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Method and kit for rapidly extracting genome DNA of gram-positive bacteria

A Gram-positive bacteria and extraction method technology, applied in the direction of recombinant DNA technology, DNA preparation, etc., can solve the problems of low efficiency and achieve the effects of high extraction throughput, a wide range of bacterial cells, and simple operation

Inactive Publication Date: 2017-02-15
北京卓诚惠生生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to overcome the defects that the current DNA extraction method is used for the extraction of Gram-positive bacterial genome DNA, and to provide a high-yield and high-purity DNA with high extraction efficiency. Kits and methods for genomic DNA of gram-positive bacteria

Method used

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  • Method and kit for rapidly extracting genome DNA of gram-positive bacteria
  • Method and kit for rapidly extracting genome DNA of gram-positive bacteria
  • Method and kit for rapidly extracting genome DNA of gram-positive bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] 1) Make materials for DNA purification according to the following formula:

[0045] Take 4 kinds of Gram-positive bacteria: Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes, and Streptococcus, pick single colonies respectively, and inoculate them in test tubes of 5 mL LB liquid medium for culture, Shake the bacteria at 37°C and 200 rpm for 8 hours until the turbidity of the bacteria is 1OD.

[0046] 2) Make materials for breaking cell walls according to the following formula:

[0047] ① DNA purification buffer: weigh 12g Chelex-100 with an electronic balance, add Chelex-100 particles to 80ml sterile water;

[0048] ②Add 1ml of 1M Tris (pH value is 9) and make the volume to 100ml;

[0049] ③Glass beads A: ≤106μm glass beads: 0.1g;

[0050] ④Glass beads B: 425μm-600μm glass beads: 0.1g.

[0051] 3) Operation steps:

[0052] (1) Take 1.0ml bacterial solution in EP tube, centrifuge at 10000g for 2min;

[0053] (2) Remove the supernatant, suspend the ...

Embodiment 2

[0091] Embodiment 2 Kit of the present invention and the commercialization kit (Tiangen biology, DP302) extraction effect comparison test on the market

[0092] Staphylococcus aureus was inoculated in a test tube of 5mL LB liquid medium for culture, and the turbidity of the bacterial solution was adjusted to 10 8 CFU / ml, serially diluted to 10 3 CFU / ml. Genomic DNA of Staphylococcus aureus was extracted according to the method of Example 1 and the method given in the instructions of the control kit (Tiangen Biology, DP302). Nano drop detection 10 8 The concentration of genomic DNA extracted from CFU / ml bacterial liquid was evaluated by fluorescence quantitative method to extract genomic DNA.

[0093] Table 2 Concentration measured by UV spectrophotometry

[0094] Reagent test kit 10 8 CFU / ml concentration

260 / 280 Kit of the present invention 89ng / μL 1.94 control kit 21ng / μL 1.81

[0095] Table 3 Fluorescent quantitative PCR method dete...

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Abstract

The invention discloses a method and a kit for rapidly extracting genome DNA of gram-positive bacteria. With the application of the method and the kit provided by the invention, the genome DNA of the gram-positive bacteria can be rapidly and effectively extracted within 10min, and meanwhile, the extracted genome DNA, without further purification, can be directly applied to molecular detection experiments. The method and the kit have the advantages of being rapid, simple and convenient, efficient, low in cost, good in university and the like; and the method and the kit are applicable to massive extraction of the genome DNA of the gram-positive bacteria.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for rapidly extracting the genome DNA of gram-positive bacteria and a kit for quickly extracting the genome DNA of gram-positive bacteria. Background technique [0002] Because the cell wall of Gram-positive bacteria is relatively thick, and the composition of the cell wall of Gram-negative bacteria is different, it brings relatively great difficulties to the field of foodborne pathogenic bacteria detection and seriously affects the detection efficiency. Among the traditional methods for extracting the genome of Gram-positive bacteria, enzymatic hydrolysis is the most commonly used method. The enzymatic method requires enzymatic digestion of the peptidoglycan component of the cell wall of Gram-positive bacteria, followed by extraction with a solvent, such as phenol / chloroform. The DNA adsorption material mainly adopts silica material, anion exchange resin and magnetic beads...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68
CPCC12N15/1006C12Q1/6806
Inventor 王雷刘晓东王晓艳张志强
Owner 北京卓诚惠生生物科技股份有限公司
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