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Bacteria genome DNA extraction liquid, preparation and application thereof

A technology of extract and genome, applied in the field of bacterial genome DNA extract and its preparation and application, can solve the problem of complex operation of bacterial genome extraction kit, ineffective release of genomic DNA, pollution of bacteria or its genomic DNA, etc. problems, to achieve the effects of reducing physical health damage, high extraction efficiency, and sufficient cracking

Active Publication Date: 2010-10-20
SHANGHAI BLOOD CENT
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, for some bacteria with special cell wall structure, such as Staphylococcus aureus, the genomic DNA cannot be released effectively, so lysozyme cannot be used for lysis
[0004] In addition, the routinely used bacterial genome extraction kit is complicated to operate, and it takes more than ten steps from collecting bacteria to obtaining genomic DNA, during which it is easy to introduce contamination from bacteria or their genomic DNA

Method used

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  • Bacteria genome DNA extraction liquid, preparation and application thereof

Examples

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Effect test

Embodiment 1

[0029] ① Add appropriate amount of SDS, NP-40 and Tween to water and mix, then filter and sterilize with a 0.22 μm filter, and finally add appropriate amount of chelex-100 to make the final concentration 5% (g / ml), adjust SDS, NP-40 and The concentration of Tween is 0.03% (g / ml), 1% (ml / ml) and 1% (ml / ml) respectively, and the mixture A solution is obtained;

[0030] ②Mix the stock solutions of lysostaphin, wall-breaking enzyme and proteinase K at a volume ratio of 1:5:1.25, filter through a YM-100 protein filter column at 14,000 g / min, and centrifuge at 4°C for 24 minutes to obtain liquid B;

[0031] ③ Add 230 μl of the liquid obtained in step ① to 100 μl of the liquid obtained in step ② to obtain the product bacterial genome DNA extract.

[0032] Follow the procedure below to extract bacterial genomic DNA:

[0033] ① Take 200 μl of platelet products, centrifuge at 13000g / min for 10 minutes, and collect bacteria;

[0034] ② Discard the supernatant, add 33 μl of the extract ...

Embodiment 2

[0040] ① Add appropriate amount of SDS, NP-40 and Tween to water and mix, then filter and sterilize with a 0.22 μm filter, and finally add appropriate amount of chelex-100 to make the concentration 10% (g / ml), SDS, NP-40 and Tween Make up concentration to be 0.05% (g / ml), 1.5% (ml / ml) and 1.5% (ml / ml) respectively, make A solution;

[0041] ②Mix the stock solutions of lysostaphin, wall-breaking enzyme and proteinase K at a ratio of 1.5:4.5:1.75, filter through a YM-100 protein filter column at 14,000 g / min, and centrifuge at 4°C for 24 minutes to obtain liquid B;

[0042] ③ Add 400 μl of the liquid obtained in step ① to 200 μl of the liquid obtained in step ② to obtain the product bacterial genome

[0043] DNA extraction solution.

[0044] Follow the procedure below to extract bacterial genomic DNA:

[0045] ① Take 800μl of plasma, centrifuge at 13000g / min for 10 minutes, and collect bacteria;

[0046] ② Discard the supernatant, add 50 μl of the extract of the present inven...

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Abstract

The invention discloses a bacteria genome DNA extracting solution in a blood and platelet product and a preparation method and an application method thereof. The bacteria genome DNA extracting solution consists of chelex-100, tween, NP-40, SDS, lysostaphin, a wall-breaking enzyme, a protease K and water. The extracting solution can be effectively used for extracting a bacteria genome DNA with high extraction efficiency. The preparation method and the application method are simple and convenient for operation.

Description

technical field [0001] The invention relates to an extraction solution and a preparation method for extracting bacterial genome DNA from blood and platelet products and a method for using the extract solution to extract bacterial genome DNA. Background technique [0002] Bacterial contamination in blood and platelet products has become a major hidden danger threatening the safety of blood products. It has received much attention in recent years. In the past, the detection of bacterial contamination was based on the traditional culture method. This method generally takes 24 hours to obtain the preliminary report and seven days to obtain the final report. For some bacteria with small amount of bacteria or difficult to culture, false negative results are easy to be produced, which endangers the safety of blood transfusion. With the development of molecular biology techniques, PCR and fluorescent quantitative PCR techniques are widely used in the diagnosis of bacteria. This n...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12N15/31
Inventor 刘晓颖马敏王迅郑岚钱开诚
Owner SHANGHAI BLOOD CENT
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