CN101319213B - Bacteria genome DNA extraction liquid, preparation and application thereof - Google Patents

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

Bacteria genome DNA extraction liquid and its production and application

Technical field

The present invention relates to a kind of from blood, platelet product extracting bacterial genomes DNA extracting solution and preparation method and use this extract to extract the method for bacterial genomes DNA.

Background technology

The bacterial contamination problem that exists in blood, the platelet product becomes a big hidden danger of danger side of body blood products safety.Enjoy attention in recent years.Detection for bacterial contamination in the past all is based on traditional cultural method, and this method length consuming time generally need draw first interim report in 24 hours, seven days acquisition final reports.And for some bacterium amount less or the bacterium that is difficult to cultivate easily produce false negative result, jeopardized the security of blood transfusion.Along with the development of Protocols in Molecular Biology, PCR, fluorescent quantitative PCR technique are widely used in the diagnosis of bacterium.This not only makes the bacterium that was difficult to originally cultivate be detected, and has shortened detection time greatly.In this operating process, bacterial genomes extracting work is most important.

The conventional commercialization bacterial genomes extraction agent box that uses based on SDS/ phenol/chloroform extraction, for improving lytic effect, adds N,O-Diacetylmuramidase usually, strengthens the lytic effect of bacteria cell wall.β-1, the 4 key molecule that N,O-Diacetylmuramidase can cut off between N-acetylglucosamine and the N-acetylmurami connects, thereby destroys the bacterial cell wall construction.But for the more special bacterium of the texture ratio of some cell wallss, as streptococcus aureus, its genomic dna can not discharge effectively, therefore can not use N,O-Diacetylmuramidase to carry out bacteriolyze.

In addition, the conventional bacterial genomes extraction agent box complicated operation that uses, it is rapid to experience ten multisteps from the collection bacterium to the acquisition genomic dna, is easy to introduce the pollution of bacterium or its genomic dna therebetween.Therefore need be a kind of quick, be applicable to the method for from blood, platelet product, extracting bacterial genomes DNA effectively.

Summary of the invention

One of purpose of the present invention just is to provide a kind of new bacteria genome DNA extraction liquid, and this kind DNA extract can make things convenient for extracting bacterial genomes DNA, and the extraction efficiency height.

Another object of the present invention provides the preparation method of above-mentioned bacteria genome DNA extraction liquid.

Another object of the present invention provides the concrete using method of above-mentioned bacteria genome DNA extraction liquid.

For reaching above-mentioned purpose, the concrete technical scheme that the present invention takes is:

A kind of bacteria genome DNA extraction liquid is that 50～70% first liquid and 30～50% second liquid are formed by volume percent, wherein

First liquid is made up of chelex-100, SDS, NP-40 and tween, and concentration is respectively 5%～20%g/ml, 0.03%～3%g/ml, 0.5%～5%V/V and 0.5%～5%V/V, and all the other are water;

Second liquid is made up of lysostaphin, wall breaking enzyme (10U/ μ l) and Proteinase K (20mg/ml), and volume ratio is 0.5～1.5: 4.5～5.5: 0.75～1.75.

In the above-mentioned second liquid, the original liquid concentration of lysostaphin is preferably 2mg/ml, wall breaking enzyme 10U/ μ l, Proteinase K 20mg/ml.

The preparation method of above-mentioned bacteria genome DNA extraction liquid comprises the following steps:

1. getting SDS, NP-40 and tween adds in the entry and mixes, filtration sterilization then, add an amount of chelex-100 at last, be made into concentration and be respectively the mixture of SDS 0.03%～3%g/ml, NP-400.5%～5%V/V, tween 0.5%～5%V/V and chelex-1005%～20%g/ml, make first liquid;

2. the stoste of lysostaphin, wall breaking enzyme and Proteinase K is mixed by 0.5～1.5: 4.5～5.5: 0.75～1.75 volume ratio, it is centrifugal that YM-100 albumen Filter column filters the back, makes second liquid;

3. in the second liquid that will 2. obtain through the first liquid adding step that 1. step obtains in proportion.

In this extract, first liquid can destroy bacteria cell wall in part degree ground, and chelex-100 closes exchanger as a kind of slightly acidic huge legendary turtle, and polyvalent cation is had very strong selectivity, can effectively remove this class and pollute the restraining effect of impurity to follow-up PCR reaction.And the adding of second liquid has promoted the release of bacterial genomes DNA by the further lytic effect that strengthens bacteria cell wall of the combined action of multiple enzyme.

Use extract of the present invention,, may further comprise the steps in order to extract the method for DNA of bacteria in blood and the platelet product:

1. get sample 50～5000 μ l, 10000～15000g/ minute, centrifugal 10～15min collected bacterium;

2. abandon supernatant liquor, add above-mentioned extract 25～250 μ l;

3. hatched 30～60 minutes for 30～37 ℃, hatched 20～120 minutes for 50～65 ℃;

4. heated and boiled is 5～20 minutes, places 5～120 seconds in 0 ℃;

5. 10000～15000g/ minute, centrifugal 10～15 minutes, the gained supernatant liquor was bacterial genomes DNA.

Advantage of the present invention and beneficial effect:

In the present invention, utilized the characteristics of bacteria cell wall and the character of DNA, created extraction bacterial genomes DNA method from blood and thrombocyte based on these characteristics and character.Compare with other conventional DNA of bacteria extracting method, the present invention has several tangible advantages and beneficial effect:

1. the extract preparation simply is convenient to use, stores, is transported, and this extract only is made up of two kinds of reagent, needs only during use it is carried out mixing in proportion; 2. extractive process is easy, the shortest bacterial genomes DNA that can be in 90 minutes extracts from sample; 3. extraction efficiency height, the bacteria cell wall cracking is abundant, and the genomic dna that discharges all is present in the supernatant liquor; 4. leaching process does not use phenol, chloroform, has reduced the healthy injury to the experimenter.

Embodiment

Embodiment 1:

Make bacterial genomes DNA extraction liquid by following prescription:

1. an amount of SDS, NP-40 and tween are added in the entry and mix, use 0.22 μ m filter filtration sterilization then, add an amount of chelex-100 at last, making ultimate density is 5% (g/ml), the concentration of regulating SDS, NP-40 and tween is respectively 0.03% (g/ml), 1% (ml/ml) and 1% (ml/ml), makes mixture first liquid;

2. with 1: 5: 1.25 by volume the mixed of stoste of lysostaphin, wall breaking enzyme and Proteinase K, YM-100 albumen Filter column filtered 14000g/ minute, 4 ℃ centrifugal 24 minutes, make second liquid;

3. the liquid 230 μ l that will 1. obtain through step add the liquid that 2. 100 μ l obtain through step, promptly get the product bacteria genome DNA extraction liquid.

Operate to extract bacterial genomes DNA by following program:

1. get platelet product 200 μ l, 13000g/ minute, centrifugal 10 minutes, collect bacterium;

2. abandon supernatant, add extract 33 μ l of the present invention,

3. hatched 30 minutes for 37 ℃, hatched 30 minutes for 56 ℃,

4. heated and boiled is 10 minutes, in 0 ℃ of placement 60 seconds,

5. 13000g/ minute, centrifugal 10 minutes, the gained supernatant liquor was bacterial genomes DNA ,-20 ℃ of preservations.

Embodiment 2:

Make bacterial genomes DNA extraction liquid by following prescription:

1. an amount of SDS, NP-40 and tween are added in the entry and mix, use 0.22 μ m filter filtration sterilization then, add an amount of chelex-100 at last, making concentration is 10% (g/ml), SDS, NP-40 and tween are made into concentration and are respectively 0.05% (g/ml), 1.5% (ml/ml) and 1.5% (ml/ml), make first liquid;

2. with the stoste of lysostaphin, wall breaking enzyme and the Proteinase K mixed by 1.5: 4.5: 1.75, YM-100 albumen Filter column filtered 14000g/ minute, 4 ℃ centrifugal 24 minutes, make second liquid;

3. the liquid 400 μ l that will 1. obtain through step add the liquid that 2. 200 μ l obtain through step, promptly get the product bacterial genomes

The DNA extract.

Operate to extract bacterial genomes DNA by following program:

1. get blood plasma 800 μ l, 13000g/ minute, centrifugal 10 minutes, collect bacterium;

2. abandon supernatant, add extract 50 μ l of the present invention,

3. hatched 50 minutes for 37 ℃, hatched 60 minutes for 56 ℃,

4. heated and boiled is 10 minutes, in 0 ℃ of placement 60 seconds,

5. 13000g/ minute, centrifugal 10 minutes, the gained supernatant liquor was bacterial genomes DNA ,-20 ℃ of preservations.

The test example

Use bacteria genome DNA extraction liquid embodiment 1 of the present invention and commercial kit TaKaRa MiniBESTBacterial Genomic DNA Extraction Kit Ver.2.0, colon bacillus and streptococcus aureus to the platelet product moderate carries out extracting respectively, use fluorescent quantitative PCR technique checking extraction efficiency subsequently, the result is as follows:

Two kinds of method extracting bacterial genomes gained CT values of quantitative fluorescent PCR checking

Use bacteria genome DNA extraction liquid embodiment 1 of the present invention and carry out extracting in two kinds of bacterium, extract product is used quantitative fluorescent PCR and is identified, its CT value is all less than the CT value of test kit method gained bacterial genomes dna profiling.Prove bacteria genome DNA extraction liquid extraction efficiency height of the present invention.

Claims (4)

1. bacteria genome DNA extraction liquid is that 50～70% first liquid and 30～50% second liquid are formed by volume percent, wherein

First liquid is made up of chelex-100, SDS, NP-40, tween and water, and concentration is respectively 5%～20%g/ml, 0.03%～3%g/ml, 0.5%～5%V/V and 0.5%～5%V/V, and all the other are water;

Second liquid is made up of lysostaphin stoste, wall breaking enzyme stoste and Proteinase K stoste, and volume ratio is 0.5～1.5: 4.5～5.5: 0.75～1.75, wherein, wall breaking enzyme original liquid concentration 10U/ μ l, Proteinase K original liquid concentration 20mg/ml.

2. bacteria genome DNA extraction liquid as claimed in claim 1 is characterized in that: in the second liquid, the original liquid concentration of lysostaphin is 2mg/ml.

3. the preparation method of claim 1 or 2 described bacteria genome DNA extraction liquids comprises the following steps:

1. getting SDS, NP-40 and tween adds in the entry and mixes, filtration sterilization then, add an amount of chelex-100 at last, be made into concentration and be respectively the mixture of SDS 0.03%～3%g/ml, NP-400.5%～5%V/V, tween 0.5%～5%V/V and chelex-1005%～20%g/ml, make first liquid;

2. the stoste of lysostaphin, wall breaking enzyme and Proteinase K is mixed by 0.5～1.5: 4.5～5.5: 0.75～1.75 volume ratio, it is centrifugal that YM-100 albumen Filter column filters the back, makes second liquid;

3. in the second liquid that will 2. obtain through the first liquid adding step that 1. step obtains in proportion.

4. use claim 1 or 2 described bacteria genome DNA extraction liquids in order to extract the method for DNA of bacteria in blood and the platelet product, may further comprise the steps:

1. get sample 50～5000 μ l, 10000～15000g/ minute, centrifugal 10～15min collected bacterium;

2. abandon supernatant liquor, add above-mentioned extract 25～250 μ l;

3. hatched 30～60 minutes for 30～37 ℃, hatched 20～120 minutes for 50～65 ℃;

4. heated and boiled is 5～20 minutes, places 5～120 seconds in 0 ℃;

5. 10000～15000g/ minute, centrifugal 10～15 minutes, the gained supernatant liquor was bacterial genomes DNA.