CN203513676U - Extraction device of bacteria DNA (Desoxvribose Nucleic Acid) in enrichment broth - Google Patents
Extraction device of bacteria DNA (Desoxvribose Nucleic Acid) in enrichment broth Download PDFInfo
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- CN203513676U CN203513676U CN201320604940.2U CN201320604940U CN203513676U CN 203513676 U CN203513676 U CN 203513676U CN 201320604940 U CN201320604940 U CN 201320604940U CN 203513676 U CN203513676 U CN 203513676U
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- Prior art keywords
- filtering
- dna
- bacteria
- tube
- sleeve pipe
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- 241000894006 Bacteria Species 0.000 title claims abstract description 38
- 238000000605 extraction Methods 0.000 title claims abstract description 19
- 150000007523 nucleic acids Chemical class 0.000 title abstract 2
- 102000039446 nucleic acids Human genes 0.000 title abstract 2
- 108020004707 nucleic acids Proteins 0.000 title abstract 2
- 238000001914 filtration Methods 0.000 claims abstract description 58
- 239000002131 composite material Substances 0.000 claims abstract description 4
- 239000003365 glass fiber Substances 0.000 claims abstract description 4
- 239000007788 liquid Substances 0.000 claims description 24
- 239000012528 membrane Substances 0.000 claims description 18
- 239000004743 Polypropylene Substances 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 5
- -1 polypropylene Polymers 0.000 claims description 5
- 229920001155 polypropylene Polymers 0.000 claims description 5
- 230000008961 swelling Effects 0.000 claims description 3
- 238000000034 method Methods 0.000 abstract description 25
- 238000005336 cracking Methods 0.000 abstract description 7
- 238000000746 purification Methods 0.000 abstract description 6
- 239000000758 substrate Substances 0.000 abstract description 5
- 235000010633 broth Nutrition 0.000 abstract 2
- 241000192125 Firmicutes Species 0.000 abstract 1
- 230000007547 defect Effects 0.000 abstract 1
- 230000000717 retained effect Effects 0.000 abstract 1
- 238000000197 pyrolysis Methods 0.000 description 6
- 108010067770 Endopeptidase K Proteins 0.000 description 3
- 230000009089 cytolysis Effects 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 239000006166 lysate Substances 0.000 description 3
- 238000005374 membrane filtration Methods 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000000703 high-speed centrifugation Methods 0.000 description 2
- 238000007654 immersion Methods 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- CHRJZRDFSQHIFI-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;styrene Chemical compound C=CC1=CC=CC=C1.C=CC1=CC=CC=C1C=C CHRJZRDFSQHIFI-UHFFFAOYSA-N 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 230000007096 poisonous effect Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- Sampling And Sample Adjustment (AREA)
Abstract
The utility model provides an extraction device of bacteria DNA (Desoxvribose Nucleic Acid) in an enrichment broth. The extraction device comprises an inner filtering tube, a first sleeve and a second sleeve, wherein a tube body of the inner filtering tube can be embedded into the first sleeve and the second sleeve; the expanded part at the top end of the inner filtering tube is retained at the openings of the first sleeve and the second sleeve; an inner tube cover is arranged at the top end of the inner filtering tube; the bottom end of the inner filtering tube is narrowed to form the opening of the inner tube; a filtering film is arranged above the opening of the inner tube; a film pressing ring for pressing the filtering film is arranged above the filtering film; a composite filtering film consisting of more than two layers of glass fiber is adopted as the filtering film; the aperture of the filtering film is 0.2-20 microns. Due to adoption of a direct film filtering cracking method, the defects of a purification method and a direct cracking method are overcome, the DNA can be extracted for one time after bacteria in a plurality of enrichment broths are enriched and mixed, and the extraction method is not only applicable to gram-negative bacteria, but also applicable to gram-positive bacteria, and is capable of effectively eliminating interference of a sample substrate.
Description
Technical field
The utility model relates to and from one or more enrichment liquids, extracts the device that PCR detects DNA of bacteria used, and described enrichment liquid is after membrane filtration, and By Direct Pyrolysis bacterium is extracted DNA.
Background technology
The extracting method of DNA of bacteria in enrichment liquid, can simply be divided into two classes by principle, method of purification and By Direct Pyrolysis method.The method of method of purification has multiple, but all follows following flow process: bacteria lysis, and---enrichment DNA---cleaning DNA---is dissolving DNA again.The advantage of the method is that the DNA purity obtaining is high, and PCR supressor content is very low; Its shortcoming is that DNA loss is more in leaching process, causes the yield of final DNA low.Operation steps is more in addition, also easily produces crossed contamination.By Direct Pyrolysis method operation steps is few, do not adopt the purification process such as enrichment, cleaning, directly will after bacteria lysis, extract DNA, therefore the loss of DNA is less, but residual PCR supressor can affect follow-up PCR reaction, and sample substrate also can detect and have a negative impact PCR.
The extracting method of DNA of bacteria in desirable enrichment liquid, should possess following characteristics: DNA purity is high, the yield of DNA is high, PCR supressor content is low, easy and simple to handle, do not use poisonous organic solvent etc.For PCR detects, after extracting method preferably can merge the Enrichment of bacteria in many parts of enrichment liquids, single extraction DNA; And method for extracting is not only applicable to Gram-negative bacteria, be also applicable to the firm gram-positive microorganism that is difficult to cracking of cell walls; And can also effectively remove the interference of sample substrate.
Utility model content
The utility model is for above-mentioned problem, provides from a or many parts of enrichment liquids and extracts the device that PCR detects DNA of bacteria used.
Specifically, the extraction element of DNA of bacteria in enrichment liquid described in the utility model, comprise and filter inner tube, the first sleeve pipe and the second sleeve pipe, filtering the body of inner tube can insert in the first sleeve pipe and the second sleeve pipe, the ultimate swelling portion of filtering inner tube stays outside the mouth of pipe of the first sleeve pipe and the second sleeve pipe, and the top of filtering inner tube has inner tube lid, and the bottom constriction of filtering inner tube is interior tube opening, above interior tube opening, be provided with filtering membrane, filtering membrane top is provided with for compressing the press mold circle of filtering membrane.
Further, described filtering membrane is the composite filter membrane that two-layer above glass fibre is made.
Further, the aperture of described filtering membrane is 0.2-20um.
Further, described the second sleeve pipe has or covers without pipe.
Further, the material of described filtration inner tube is polypropylene.
Further, the material of described the first sleeve pipe and the second sleeve pipe is polypropylene.
In enrichment liquid described in the utility model, the using method of the extraction element of DNA of bacteria is as described below:
1, concentrated bacterium: in filtering inner tube, add enrichment liquid, with centrifugal 1 minute of 1000xg centrifugal force (or filtering under 10mmHg-15mmHg pressure 1 minute), then with centrifugal 1 minute of 6000xg centrifugal force (or filtering under 20mmHg-30mmHg pressure 1 minute);
2, remove lipid: in filtering inner tube, add 75% alcohol immersion 1 minute, with centrifugal 1 minute of 6000xg centrifugal force (or filtering 1 minute) under 20mmHg-30mmHg pressure;
3, cracking bacterium: the outer tube more renewing, filtration inner tube is inserted in the second sleeve pipe.In filtering inner tube, add EB lysate (containing Proteinase K), hatch 30 minutes (gram positive bacterium extends to 60 minutes) for 55 ℃;
4, heat inactivation: hatch 10 minutes inactivated proteases K for 95 ℃; With centrifugal 3 minutes of 12000xg centrifugal force (or filtering under 20mmHg-30mmHg pressure 1 minute), the solution in the second sleeve pipe is the DNA of bacteria solution detecting for PCR.
Device described in the utility model, adopt membrane filtration By Direct Pyrolysis method, the shortcoming of having evaded method of purification and By Direct Pyrolysis method, can the bacterium in many parts of enrichment liquids be merged, only need DNA extracting flow process one time, and method for extracting is not only applicable to Gram-negative bacteria, be also applicable to gram-positive microorganism; And can also effectively remove the interference of sample substrate.There is following features:
1, with membrane filtration, substituted the concentrated bacterium of high speed centrifugation, omitted the step that after high speed centrifugation, supernatant liquor is abandoned in suction from centrifuge tube, operate easylier, and can effectively avoid crossed contamination and Biosafety risk, and can be to carrying out in the lump DNA extracting after the Enrichment of bacteria in a plurality of enrichment liquids;
2, increase the step of enrichment liquid degrease, can effectively remove the fat-soluble component of sample and the substratum PCR inhibitor in residual, eliminated matrix interference;
3, special crack liquid formula and low temperature pyrolyzer mode, be not only applicable to the extraction of Gram-negative bacteria DNA, and be applicable to the extraction of the gram positive microbes DNA of more difficult cracking, when improving lysis efficiency, reduced fracture and the degraded of DNA.
Accompanying drawing explanation
Fig. 1 filters the schematic diagram that inner tube is inserted the first sleeve pipe in the utility model embodiment;
Fig. 2 filters the schematic diagram that inner tube is inserted the second sleeve pipe in the utility model embodiment;
Wherein, 1 be the first sleeve pipe, 2 for filter inner tube, 20 for inner tube lid, 21 for interior tube opening, 3 for press mold circle, 4 for filtering membrane, 5 be that the second sleeve pipe, 50 is that the second casing pipe covers.
Embodiment
Below in conjunction with the drawings and specific embodiments, the extraction element of DNA of bacteria in enrichment liquid described in the utility model to be described without limitation, object is that the public understands described technical scheme better.
As shown in Figure 1-2, the extraction element of DNA of bacteria in enrichment liquid described in the utility model, comprise and filter inner tube 2, the first sleeve pipe 1 and the second sleeve pipe 5, filtering the body of inner tube 2 can insert in the first sleeve pipe 1 and the second sleeve pipe 5, the ultimate swelling portion of filtering inner tube 2 stays outside the mouth of pipe of the first sleeve pipe 1 and the second sleeve pipe 5, the top of filtering inner tube 2 has inner tube lid 20, the bottom constriction of filtering inner tube 2 is interior tube opening 21, above interior tube opening 21, be provided with filtering membrane 4, filtering membrane 4 tops are provided with for compressing the press mold circle 3 of filtering membrane 4, the composite filter membrane that described filtering membrane 4 is made for two-layer above glass fibre, aperture is 0.2-20um, the second sleeve pipe 5 has or nothing the second casing cover 50, described filtration inner tube, the material of the first sleeve pipe and the second sleeve pipe is polypropylene.
The using method of device described in the utility model is as follows:
1, concentrated bacterium: add 200ul-400ul enrichment liquid in filtering inner tube, with centrifugal 1 minute of 1000xg centrifugal force (or filtering under 10mmHg-15mmHg pressure 1 minute), then with centrifugal 1 minute of 6000xg centrifugal force (or filtering under 20mmHg-30mmHg pressure 1 minute).
2, remove lipid: in filtering inner tube, add 200ul75% alcohol immersion 1 minute, with centrifugal 1 minute of 6000xg centrifugal force (or filtering 1 minute) under 20mmHg-30mmHg pressure.
3, cracking bacterium: the outer tube more renewing, filtration inner tube is inserted in the second sleeve pipe.In filtering inner tube, add 200ul EB lysate (containing Proteinase K), hatch 30 minutes (gram positive bacterium extends to 60 minutes) for 55 ℃.
4, heat inactivation: hatch 10 minutes inactivated proteases K for 95 ℃; With centrifugal 3 minutes of 12000xg centrifugal force (or filtering under 20mmHg-30mmHg pressure 1 minute), the solution in outer tube is the DNA of bacteria solution detecting for PCR.
Wherein, EB lysate formula is: deionized water, vinylbenzene-divinylbenzene polymer 5-20%, Proteinase K 0.05-1mg/ml(add before using); Cracking incubation temperature: 55 ℃.
Apply the method for device described in the utility model, evaded the shortcoming of method of purification and By Direct Pyrolysis method, after merging the Enrichment of bacteria in many parts of enrichment liquids, single extraction DNA, and the interference that can also effectively remove sample substrate.
Claims (6)
1. the extraction element of DNA of bacteria in an enrichment liquid, it is characterized in that: comprise and filter inner tube, the first sleeve pipe and the second sleeve pipe, filtering the body of inner tube can insert in the first sleeve pipe and the second sleeve pipe, the ultimate swelling portion of filtering inner tube stays outside the mouth of pipe of the first sleeve pipe and the second sleeve pipe, the top of filtering inner tube has inner tube lid, the bottom constriction of filtering inner tube is interior tube opening, above interior tube opening, is provided with filtering membrane, and filtering membrane top is provided with for compressing the press mold circle of filtering membrane.
2. the extraction element of DNA of bacteria in enrichment liquid according to claim 1, is characterized in that: described filtering membrane is the composite filter membrane that two-layer above glass fibre is made.
3. the extraction element of DNA of bacteria in enrichment liquid according to claim 1 and 2, is characterized in that: the aperture of described filtering membrane is 0.2-20um.
4. the extraction element of DNA of bacteria in enrichment liquid according to claim 1 and 2, is characterized in that: described the second sleeve pipe has or covers without pipe.
5. the extraction element of DNA of bacteria in enrichment liquid according to claim 1 and 2, is characterized in that: the material of described filtration inner tube is polypropylene.
6. the extraction element of DNA of bacteria in enrichment liquid according to claim 1 and 2, is characterized in that: the material of described the first sleeve pipe and the second sleeve pipe is polypropylene.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201320604940.2U CN203513676U (en) | 2013-09-27 | 2013-09-27 | Extraction device of bacteria DNA (Desoxvribose Nucleic Acid) in enrichment broth |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201320604940.2U CN203513676U (en) | 2013-09-27 | 2013-09-27 | Extraction device of bacteria DNA (Desoxvribose Nucleic Acid) in enrichment broth |
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| Publication Number | Publication Date |
|---|---|
| CN203513676U true CN203513676U (en) | 2014-04-02 |
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| CN201320604940.2U Expired - Fee Related CN203513676U (en) | 2013-09-27 | 2013-09-27 | Extraction device of bacteria DNA (Desoxvribose Nucleic Acid) in enrichment broth |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105910863A (en) * | 2016-04-18 | 2016-08-31 | 佛山盈诺科创投资咨询中心（有限合伙） | A body fluid enrichment smear machine |
| WO2017201046A1 (en) * | 2016-05-17 | 2017-11-23 | Integrated Nano-Technologies, Inc. | Filtration column assembly for diagnostic assay system |
| CN110724633A (en) * | 2019-11-11 | 2020-01-24 | 浙江汇泽医药科技有限公司 | Trace cell nucleic acid extraction and amplification system and process |
| WO2020211637A1 (en) * | 2019-04-19 | 2020-10-22 | 安徽森芃生物科技有限责任公司 | Method for purifying and concentrating dna in forensic samples using selective filtration column |
| CN112683990A (en) * | 2021-03-11 | 2021-04-20 | 中国疾病预防控制中心传染病预防控制所 | Biological safety pretreatment method for microorganism identification sample of MALDI-TOF MS |
-
2013
- 2013-09-27 CN CN201320604940.2U patent/CN203513676U/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105910863A (en) * | 2016-04-18 | 2016-08-31 | 佛山盈诺科创投资咨询中心（有限合伙） | A body fluid enrichment smear machine |
| CN105910863B (en) * | 2016-04-18 | 2019-08-09 | 佛山盈诺科创投资咨询中心（有限合伙） | Body fluid enrichment smear machine |
| WO2017201046A1 (en) * | 2016-05-17 | 2017-11-23 | Integrated Nano-Technologies, Inc. | Filtration column assembly for diagnostic assay system |
| US11148135B2 (en) | 2016-05-17 | 2021-10-19 | Integrated Nano-Technologies, Inc. | Filtration column assembly for diagnostic assay system |
| WO2020211637A1 (en) * | 2019-04-19 | 2020-10-22 | 安徽森芃生物科技有限责任公司 | Method for purifying and concentrating dna in forensic samples using selective filtration column |
| CN110724633A (en) * | 2019-11-11 | 2020-01-24 | 浙江汇泽医药科技有限公司 | Trace cell nucleic acid extraction and amplification system and process |
| CN112683990A (en) * | 2021-03-11 | 2021-04-20 | 中国疾病预防控制中心传染病预防控制所 | Biological safety pretreatment method for microorganism identification sample of MALDI-TOF MS |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140402 Termination date: 20200927 |