CN106381294A - Extraction method for microbial DNA in milk - Google Patents
Extraction method for microbial DNA in milk Download PDFInfo
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- CN106381294A CN106381294A CN201510474702.8A CN201510474702A CN106381294A CN 106381294 A CN106381294 A CN 106381294A CN 201510474702 A CN201510474702 A CN 201510474702A CN 106381294 A CN106381294 A CN 106381294A
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Abstract
The invention discloses an extraction method for microbial DNA in milk. The method comprises the following steps: adding 0.8 mL of a milk sample into 0.3 to 0.5 mL of an ES solution (EDTA with a pH value of 7.5 and concentration of 0.5 mol/L and SDS with concentration of 2%), carrying out uniform mixing and centrifugation and discarding supernatant; adding 0.8 to 1.2 mL of a PBS buffer solution, carrying out oscillation and shaking up, then carrying out centrifugation and discarding supernatant; adding 20 to 30 [mu]L of Staphylococcus lysozyme with a mass concentration of 0.02 mg/mL; adding 150 to 200 [mu]L of an aqueous Chelex-100 solution with a mass concentration of 10%, carrying out violent oscillation for 5 to 10 s and then carrying out heating in boiling water with a temperature of 100 DEG C for 8 to 12 min; and carrying out centrifugation and taking supernatant for a PCR reaction. The method provided by the invention employs a Chelex-100 process and can economically, rapidly, efficiently and directly extract bacterial DNA from the milk sample; and the bacterial DNA can be used for PCR detection of main pathogenic bacteria causing mastitis in cow.
Description
Technical field
The present invention relates to milch cow technical field of molecular biology, the extracting method of microbial DNA in specifically a kind of milk.
Background technology
Mammitis of cow is one of most important disease of impact China or even developing of world milk industry, staphylococcus aureuses, no
The infection of the pathogenic bacterium such as Streptococcus lactis (Lister) Lohnis 1909.554., Mycoplasma bovis and escherichia coli is the main reason causing this disease.At present, domestic still
Lack effective vaccine for preventing mammitis of cow.Prevent and treat this sick key factor to be to identify its pathogenic bacterium, once it is determined that
Pathogenic bacterium, then can take appropriate measures immediately.
For pathogens from dairy cattle affected smastitis PCR detect in, in milk sample the preparation of DNA of bacteria template be impact detection speed,
One of principal element of sensitivity.For shortening detection time, in domestic extracting directly milk, the method for DNA of pathogenic has at present：
1. phenol chloroform method, its operating procedure is complex, time-consuming, loss DNA while, also easily cause each milk sample it
Between pollution；Remain in the Organic substances such as the phenol in DNA has certain inhibitory action to archaeal dna polymerase.Additionally, this method needs corruption
Corrosion, poisonous organic solvent, such as phenol etc., there is harmful effect to personnel, environment.2. nucleic acid purification post method, due to
During its complexity and higher cost are making it difficult to apply to the detection of high-volume milk sample.3. paramagnetic particle method, this method extracts milk sample
Relatively costly needed for DNA of bacteria, limit its application in a large amount of milk samples detect.
Content of the invention
It is an object of the invention to provide in a kind of milk microbial DNA extracting method, can be through using Chelex-100 method
Ji, quickly and efficiently extracting directly DNA of bacteria from milk sample, detect main pathogen of bovine mastitis for PCR.
For achieving the above object, the present invention provides following technical scheme：
In a kind of milk, the extracting method of microbial DNA, comprises the following steps：
(1) milk sample 0.8mL is taken to be added to 0.3～0.5mL ES solution (0.5mol/L EDTA (pH7.5), 0.2%SDS)
In, mix, then under the rotating speed of 10000～15000r/min, be centrifuged 8～12min, abandon supernatant；
(2) add 0.8～1.2mL PBS, vibration shakes up, then under the rotating speed of 8000～12000r/min from
The heart 3～8min, abandons supernatant；
(3) add the staphylococcus lysozyme that 20～30 μ L mass concentrations are 0.02mg/mL；Add 150～200 μ L mass
Percentage concentration is 10% Chelex-100 aqueous solution, acutely vibrates 5～10s, is placed in heating 8～12min in 100 DEG C of boiling water；
Then it is centrifuged 3～8min under the rotating speed of 10000～15000r/min, finally take supernatant to be used for PCR and react.
As the further scheme of the present invention：In described step (1), the consumption of ES solution is 0.4mL.
As the further scheme of the present invention：In described step (1), rotating speed is 12000r/min, and centrifugation time is 10min.
As the further scheme of the present invention：In described step (2), the consumption of PBS is 1mL.
As the further scheme of the present invention：In described step (2), rotating speed is 10000r/min, and centrifugation time is 5min.
As the further scheme of the present invention：In described step (3), the consumption of staphylococcus lysozyme is 25 μ L, Chelex-100
The consumption of aqueous solution is 175 μ L.
As the further scheme of the present invention：In described step (3), in boiling water, heat 10min.
As the further scheme of the present invention：In described step (3), rotating speed is 12000r/min, and centrifugation time is 5min.
Explain below for above-mentioned technical term：
Chelex-100 is that one kind is negatively charged in neutral and alkaline conditions, water-fast chelating resin, can chelate big
Amount bivalent metal ion.
Chelating resin energy adsorbing metal ions, there is complexation reaction with metal ion in the function atom on resin, form class
Rock-steady structure like small molecule chelate.
PCR is polymerase chain reaction, refers to, under archaeal dna polymerase is catalyzed, with fundamental chain DNA as template, draw with specific
Thing is to extend starting point, and by steps such as degeneration, annealing, extensions, replication in vitro goes out subchain DNA complementary with fundamental chain template DNA
Process.
Multiplex PCR adds 2 to above primer in same PCR system, can expand two or more nucleic acid fragment simultaneously,
For detecting while multiple pathogens.
Chelex-100 is that one kind is negatively charged in neutral and alkaline conditions, water-fast chelating resin, can chelate a large amount of two
Valence metal ion, therefore can be with the Protecting gene group DNA destruction from bivalent metal ion under the high temperature conditions, meanwhile, can
Ca in capture milk sample effectively2+Deng PCR mortifier, thus improving the sensitivity of follow-up PCR reaction；Chelex-100 hangs
Liquid can make bacteria lysis when boiling, and so that DNA is discharged.Chelex-100 method extracts DNA, without protease K digesting,
Without decomposition agent or column purification system, the Chelex-100 granule after chelated metal ions can be removed by centrifugation, does not affect
Subsequently need Mg2+PCR reaction.
Compared with prior art, the invention has the beneficial effects as follows：The present invention adopt Chelex-100 method can economical, quick,
Effective extracting directly DNA of bacteria from milk sample, detects main pathogen of bovine mastitis for PCR.Using Chelex-100
Method extracts milk sample L-form staphylococcus aureus, no matter artificial challenge's staphylococcus aureuses milk sample, is also non-golden yellow Fructus Vitis viniferae
Coccus mastitiss milk sample, or mastitiss case milk sample, this method has all reached 100% recall rate.
Specific embodiment
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described, shows
So, described embodiment is only a part of embodiment of the present invention, rather than whole embodiments.Based in the present invention
Embodiment, the every other embodiment that those of ordinary skill in the art are obtained under the premise of not making creative work, all
Belong to the scope of protection of the invention.
Embodiment 1
In the embodiment of the present invention, in a kind of milk, the extracting method of microbial DNA, comprises the following steps：
(1) milk sample 0.8mL is taken to be added in 0.4mL ES solution (0.5mol/L EDTA (pH7.5), 0.2%SDS),
Mix, then under the rotating speed of 12000r/min, be centrifuged 10min, abandon supernatant；
(2) add 1mL PBS, vibration shakes up, and is then centrifuged 5min under the rotating speed of 10000r/min, abandons
Clearly；
(3) add the staphylococcus lysozyme that 25 μ L mass concentrations are 0.02mg/mL；Add 175 μ L mass percentage concentration
Chelex-100 aqueous solution for 10%, acutely vibrates 8s, is placed in heating 10min in 100 DEG C of boiling water；Then in 12000r/min
Rotating speed under be centrifuged 5min, finally take supernatant to be used for PCR and react.
Embodiment 2
In the embodiment of the present invention, in a kind of milk, the extracting method of microbial DNA, comprises the following steps：
(1) milk sample 0.8mL is taken to be added in 0.3mL ES solution (0.5mol/L EDTA (pH7.5), 0.2%SDS),
Mix, then under the rotating speed of 10000r/min, be centrifuged 12min, abandon supernatant；Described ES solution contains the EDTA of 0.5mol/L
With the SDS of 0.2wt%, the pH value of described EDTA is 7.5；
(2) add 0.8mL PBS, vibration shakes up, and is then centrifuged 8min under the rotating speed of 8000r/min, abandons
Clearly；
(3) add the staphylococcus lysozyme that 20 μ L mass concentrations are 0.02mg/mL；Add 150 μ L mass percentage concentration
Chelex-100 aqueous solution for 10%, acutely vibrates 5s, is placed in heating 8min in 100 DEG C of boiling water；Then in 10000r/min
Rotating speed under be centrifuged 8min, finally take supernatant to be used for PCR and react.
Embodiment 3
In the embodiment of the present invention, in a kind of milk, the extracting method of microbial DNA, comprises the following steps：
(1) milk sample 0.8mL is taken to be added in 0.5mL ES solution (0.5mol/L EDTA (pH7.5), 0.2%SDS),
Mix, then under the rotating speed of 15000r/min, be centrifuged 8min, abandon supernatant；Described ES solution contains the EDTA of 0.5mol/L
With the SDS of 0.2wt%, the pH value of described EDTA is 7.5；
(2) add 1.2mL PBS, vibration shakes up, and is then centrifuged 3min under the rotating speed of 12000r/min, abandons
Supernatant；
(3) add the staphylococcus lysozyme that 30 μ L mass concentrations are 0.02mg/mL；Add 1200 μ L mass percentage concentration
Chelex-100 aqueous solution for 10%, acutely vibrates 10s, is placed in heating 12min in 100 DEG C of boiling water；Then in 15000r/min
Rotating speed under be centrifuged 3min, finally take supernatant to be used for PCR and react.
The present invention adopts Chelex-100 method economical, quick, effective can obtain extracting directly DNA of bacteria from milk sample, is used for
PCR detects main pathogen of bovine mastitis.Milk sample L-form staphylococcus aureus are extracted using Chelex-100 method no matter
Artificial challenge's staphylococcus aureuses milk sample, is also non-staphylococcus aureus mastitis milk sample, or mastitiss case milk sample,
This method has all reached 100% recall rate.
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, and do not carrying on the back
In the case of the spirit or essential attributes of the present invention, the present invention can be realized in other specific forms.Therefore, no matter from
From the point of view of which point, embodiment all should be regarded as exemplary, and be nonrestrictive, the scope of the present invention is by appended power
Profit requires rather than described above limits, it is intended that all in the implication and scope of the equivalency of claim by falling
Change is included in the present invention.
Moreover, it will be appreciated that although this specification is been described by according to embodiment, not each embodiment only comprises
One independent technical scheme, only for clarity, those skilled in the art should be by for this narrating mode of description
As an entirety, the technical scheme in each embodiment can also be through appropriately combined, and forming those skilled in the art can for description
With the other embodiment understanding.
Claims (8)
1. in a kind of milk the extracting method of microbial DNA it is characterised in that comprising the following steps：
(1) take milk sample 0.8mL to be added in 0.3～0.5mL ES solution, mix, then in 10000～15000r/min
Rotating speed under be centrifuged 8～12min, abandon supernatant；Described ES solution contains the SDS of EDTA and 0.2wt% of 0.5mol/L,
The pH value of described EDTA is 7.5；
(2) add 0.8～1.2mL PBS, vibration shakes up, then under the rotating speed of 8000～12000r/min from
The heart 3～8min, abandons supernatant；
(3) add the staphylococcus lysozyme that 20～30 μ L mass concentrations are 0.02mg/mL；Add 150～200 μ L mass
Percentage concentration is 10% Chelex-100 aqueous solution, acutely vibrates 5～10s, is placed in heating 8～12min in 100 DEG C of boiling water；
Then it is centrifuged 3～8min under the rotating speed of 10000～15000r/min, finally take supernatant to be used for PCR and react.
2. in milk according to claim 1 the extracting method of microbial DNA it is characterised in that described step (1)
In, the consumption of ES solution is 0.4mL.
3. in milk according to claim 1 the extracting method of microbial DNA it is characterised in that described step (1)
In, rotating speed is 12000r/min, and centrifugation time is 10min.
4. in milk according to claim 1 the extracting method of microbial DNA it is characterised in that described step (2)
In, the consumption of PBS is 1mL.
5. in milk according to claim 1 the extracting method of microbial DNA it is characterised in that described step (2)
In, rotating speed is 10000r/min, and centrifugation time is 5min.
6. in milk according to claim 1 the extracting method of microbial DNA it is characterised in that described step (3)
In, the consumption of staphylococcus lysozyme is 25 μ L, and the consumption of Chelex-100 aqueous solution is 175 μ L.
7. in milk according to claim 1 the extracting method of microbial DNA it is characterised in that described step (3)
In, heat 10min in boiling water.
8. in milk according to claim 1 the extracting method of microbial DNA it is characterised in that described step (3)
In, rotating speed is 12000r/min, and centrifugation time is 5min.
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| CN201510474702.8A CN106381294A (en) | 2015-08-06 | 2015-08-06 | Extraction method for microbial DNA in milk |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108396025A (en) * | 2018-01-23 | 2018-08-14 | 安徽微分基因科技有限公司 | A method of extracting microorganism MetaDNA from lacto's sample |
| CN110527681A (en) * | 2018-05-25 | 2019-12-03 | 中国农业科学院北京畜牧兽医研究所 | The extracting method of total microbial DNA in a kind of milk |
| CN110791568A (en) * | 2018-08-03 | 2020-02-14 | 上海市质量监督检验技术研究院 | LAMP primer group for rapidly detecting chicken-derived components in beef and mutton, detection kit, detection method and application |
| CN111187768A (en) * | 2020-03-17 | 2020-05-22 | 广西壮族自治区水牛研究所 | Kit for extracting total DNA of microorganisms in buffalo milk and extraction method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101319213A (en) * | 2007-06-07 | 2008-12-10 | 上海市血液中心 | Bacteria genome DNA extraction liquid, preparation and application thereof |
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2015
- 2015-08-06 CN CN201510474702.8A patent/CN106381294A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101319213A (en) * | 2007-06-07 | 2008-12-10 | 上海市血液中心 | Bacteria genome DNA extraction liquid, preparation and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 许金朋等: "Chelex-100法直接提取乳样中奶牛乳房炎主要致病菌DNA方法的建立", 《中国畜牧兽医》 * |
| 钱雪琴等: "Chelex-100法和碱性裂解法提取细菌DNA的比较", 《中国卫生检验杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108396025A (en) * | 2018-01-23 | 2018-08-14 | 安徽微分基因科技有限公司 | A method of extracting microorganism MetaDNA from lacto's sample |
| CN110527681A (en) * | 2018-05-25 | 2019-12-03 | 中国农业科学院北京畜牧兽医研究所 | The extracting method of total microbial DNA in a kind of milk |
| CN110791568A (en) * | 2018-08-03 | 2020-02-14 | 上海市质量监督检验技术研究院 | LAMP primer group for rapidly detecting chicken-derived components in beef and mutton, detection kit, detection method and application |
| CN111187768A (en) * | 2020-03-17 | 2020-05-22 | 广西壮族自治区水牛研究所 | Kit for extracting total DNA of microorganisms in buffalo milk and extraction method and application thereof |
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