CN105695451A - Extraction method of microbial total DNA in human stomach - Google Patents
Extraction method of microbial total DNA in human stomach Download PDFInfo
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- 210000002784 stomach Anatomy 0.000 title claims abstract description 44
- 238000000605 extraction Methods 0.000 title claims abstract description 16
- 230000000813 microbial effect Effects 0.000 title abstract description 12
- 238000000034 method Methods 0.000 claims abstract description 27
- 244000005700 microbiome Species 0.000 claims description 37
- 238000005119 centrifugation Methods 0.000 claims description 27
- 239000000872 buffer Substances 0.000 claims description 22
- 239000007790 solid phase Substances 0.000 claims description 21
- 238000003752 polymerase chain reaction Methods 0.000 claims description 16
- 238000011010 flushing procedure Methods 0.000 claims description 13
- 239000006228 supernatant Substances 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 8
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- 230000001476 alcoholic effect Effects 0.000 claims description 7
- 239000007791 liquid phase Substances 0.000 claims description 7
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 6
- 239000000463 material Substances 0.000 claims description 6
- 238000007400 DNA extraction Methods 0.000 claims description 5
- 230000003321 amplification Effects 0.000 claims description 5
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 5
- 238000012408 PCR amplification Methods 0.000 claims description 4
- 239000011543 agarose gel Substances 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 3
- 239000001103 potassium chloride Substances 0.000 claims description 3
- 235000011164 potassium chloride Nutrition 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 238000001962 electrophoresis Methods 0.000 claims description 2
- 238000000870 ultraviolet spectroscopy Methods 0.000 claims description 2
- 239000000284 extract Substances 0.000 abstract description 6
- 230000000694 effects Effects 0.000 abstract description 4
- 210000004211 gastric acid Anatomy 0.000 description 7
- 230000000052 comparative effect Effects 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 238000000246 agarose gel electrophoresis Methods 0.000 description 3
- 210000004877 mucosa Anatomy 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000037149 energy metabolism Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000001156 gastric mucosa Anatomy 0.000 description 1
- 230000007661 gastrointestinal function Effects 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
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Abstract
The invention discloses an extraction method of microbial total DNA in human stomach. The extraction method includes that on the basis of an existing extraction method, a step of pre-treating stomach extract is added, so that the problems of low extraction efficiency and poor effect caused by sample low pH factors and human genome DNA interference are solved effectively. Compared with the existing method, the extraction method has the advantages that extraction efficiency is improved by one than one time, extracted microbial DNA is high in concentration, microbial total DNA is high in occupation ratio, and PCR products of 16SrRNA v1-v2 areas are bright and clear in band, so that microbial conditions in the stomach can be reflected more accurately and more comprehensively, and a foundation is laid for subsequent studying of microbial community structure and function.
Description
Technical field
The present invention relates to the extracting method of microorganism total DNA in a kind of human stomach, belong to biological technology field。
Background technology
Stomach is the pith of digestion, owing to the secretion of gastric acid makes its microorganism comprised become a part special in digestive tract microbial ecosystem, microbiologic population complicated in stomach and the health of metabolite and human body thereof and disease close relation, play an important role in human energy metabolism, alimentation, immune system, gastrointestinal function etc.。Under one's belt in the research process of the structure of community of microorganism, the extraction of microorganism total DNA is particularly important。The special environment of and low pH relatively low yet with bacterial abundance in stomach, causes microorganism total DNA extraction efficiency in stomach low, and extraction effect is poor。Common extracting mode adopts mucosa tissue as experiment sample, coordinates microbial DNA to extract test kit and extracts, and inefficiency, and the human genome DNA that major part is mucosa tissue, few part is the DNA of microorganism。So extract the DNA of gained carry out the research of follow-up molecular ecology can not biological community structure and function situation in comprehensive and accurate reaction stomach。Therefore a kind of can effectively solve the problem that sample low pH factor and human genome DNA bring the exploitation of the stomach microorganism total DNA extracting method of interference necessary。
Summary of the invention
Goal of the invention: the technical problem to be solved is to provide in a kind of human stomach the extracting method of microorganism total DNA, and the method can be greatly improved total DNA extraction efficiency and the quality of microorganism in stomach。
For solving above-mentioned technical problem, the technical solution adopted in the present invention is:
In a kind of human stomach, the extracting method of microorganism total DNA, comprises the steps:
Step 1, mixes isopyknic stomach flushing liquor and alcoholic solution, stands a period of time at-20 DEG C, by the material centrifugation at 4 DEG C after standing, removes supernatant, obtain solid phase A after centrifugation；
Step 2, adds the desired amount of buffer in the solid phase A of step 1, and mixed material carries out after mix homogeneously first time centrifugation at 4 DEG C, removes supernatant, retain solid phase after centrifugation；The solid phase of gained is carried out at 4 DEG C second time centrifugation, removes supernatant after centrifugation, obtain solid phase B；
Step 3, is sequentially added into the desired amount of E.C. 3.4.21.64 and buffer in the solid phase B of step 2, carries out heating in water bath, obtain liquid phase C after heating after mix homogeneously at 56 DEG C；
Step 4, uses DNA extraction kit to carry out the extraction of STb gene the liquid phase C of step 3, obtains STb gene, re-use micro-ultraviolet-visible spectrophotometer, carry out the mensuration of DNA total content；
Step 5, polymerase chain reaction (PCR) amplification: pcr amplification 16SrRNAv1-v2 district, the PCR primer obtained adopts agarose gel to carry out electrophoresis detection, finally gives the quality of microorganism total DNA。
Wherein, in step 1, the addition volume of described stomach flushing liquor is 10～20mL, and the addition volume of described alcoholic solution is 10～20mL, and wherein, the mass percentage concentration of described alcoholic solution is 100%。
Wherein, in step 1, the rotating speed of described centrifugation is 15000 revs/min, and centrifugation time is 10～15 minutes。
Wherein, in step 2, described buffer is made up of the component of following quality: in every 1L buffer, containing 137mmol sodium chloride, 2.7mmol potassium chloride, 10mmol dipotassium hydrogen phosphate and 2mmol potassium dihydrogen phosphate。
Wherein, in step 2, the addition of described buffer is 10mL。
Wherein, in step 2, the rotating speed of described first time centrifugation is 15000 revs/min, and centrifugation time is 10～15 minutes；The rotating speed of described second time centrifugation is 15000 revs/min, and centrifugation time is 5 minutes。
Wherein, in step 3, the addition of described E.C. 3.4.21.64 is 20 μ L, and the addition of described buffer is 200 μ L。
Wherein, in step 3, the time of described heating in water bath is 60 minutes。
Wherein, in step 5,16SrRNAv1-v2 primer is:
27F-5 '-GCCTTGCCAGCCCGCTCAGTCAGAGTTTGATCCTGGCTCAG-3 ' and
338R-5’GCCTCCCTCGCGCCATCAGNNNNNNNNCATGCTGCCTCCCGTAGGAGT-3’。
Wherein, in step 5, the mass percentage concentration of described agarose gel is 1%。
Beneficial effect: extract DNA method compared to existing gastric mucosa, the present invention extracts the method for microorganism total DNA in stomach and efficiently solves the low problem with weak effect of extraction efficiency owing to sample low pH factor and human genome DNA disturb and cause, its extraction efficiency is more than doubled, the microbial DNA concentration extracted is high, microorganism total DNA occupies ratio height, 16SrRNAv1-v2 district PCR primer band is limpid in sight, and then more accurately can react the microbial status in stomach comprehensively, the research for follow-up biological community structure Yu function is laid a good foundation。
Accompanying drawing explanation
The PCR primer band contrast effect figure that Fig. 1 is the embodiment of the present invention 1 and comparative example obtains。
Detailed description of the invention
Below in conjunction with accompanying drawing, technical scheme is described further, but the scope of protection of present invention is not limited thereto。
Embodiment 1
The extracting method of microorganism total DNA in stomach of the present invention, specifically includes following steps:
Step 1, by the stomach flushing liquor of gained in 15mL clinic and the mixing of alcoholic solution that 15mL mass percentage concentration is 100%, 24h is preserved at-20 DEG C, material after preserving is placed in constant speed centrifuge at 4 DEG C, adopt the centrifugal 10min of rotating speed of 15000 revs/min, remove supernatant after centrifugal, obtain solid phase A；
Step 2,10mL buffer is added (if the addition of amount of buffer is too low in solid phase A, the low pH that the bring interference of gastric acid then can not be completely eliminated, if the addition of amount of buffer is too high, more microorganism is then had to remain in abandoned in liquid phase, affect the extraction efficiency of DNA), use agitator vibration 2min mix homogeneously, after mix homogeneously, mixed material is placed in constant speed centrifuge at 4 DEG C, adopt the centrifugal 10min of rotating speed of 15000 revs/min, remove supernatant after centrifugation, retain solid phase；Continue the solid phase of gained to be placed in constant speed centrifuge at 4 DEG C, adopt the centrifugal 5min of rotating speed of 15000 revs/min, remove supernatant after centrifugation, obtain solid phase B；Two times centrifugal removes supernatant, eliminates the interference of gastric acid in stomach flushing liquor further；
Step 3, is sequentially added into 20 μ L E.C. 3.4.21.64s and 200 μ L buffer in solid phase B, uses agitator vibration 2min mix homogeneously, and mix homogeneously is placed in 56 DEG C of water-baths and heats 60 minutes, it is ensured that E.C. 3.4.21.64 fully reacts, and obtains liquid phase C；
Step 4, uses QIAampDNAMiniKit test kit to carry out the extraction of microorganism total DNA liquid phase C, obtains STb gene；Re-use Nanodrop2000, carry out the mensuration of DNA content。
Step 5, polymerase chain reaction (PCR) amplification: pcr amplification 16SrRNAv1-v2 district, the agarose gel electrophoresis detection that the PCR primer obtained uses mass percentage concentration to be 1%, to get rid of human genome DNA interference, it is determined that the quality of microorganism total DNA；Wherein, 16SrRNAv1-v2 primer is:
27F-5 '-GCCTTGCCAGCCCGCTCAGTCAGAGTTTGATCCTGGCTCAG-3 ' and
338R-5’GCCTCCCTCGCGCCATCAGNNNNNNNNCATGCTGCCTCCCGTAGGAGT-3’。
The addition volume of extracting method stomach flushing liquor of the present invention is 10～20mL, after pretreatment, gained solid phase sample is DNA extraction kit the best applied sample amount, stomach flushing liquor is lower than 10mL, then carried DNA concentration is relatively low, do not meet follow-up test demand, higher than 20mL, then DNA concentration will not improve therewith, adds the impact of gastric acid on the contrary。
In extracting method of the present invention, the formula of used buffer is: in every 1L buffer, containing 137mmol sodium chloride, 2.7mmol potassium chloride, 10mmol dipotassium hydrogen phosphate and 2mmol potassium dihydrogen phosphate。When using this buffer, concentrated hydrochloric acid is adopted to regulate the pH value of buffer to 7.4。Add appropriate buffer in the sample, it is possible to overcome the impact of gastric acid in sample, and to make the pH of system be about 7。
The concentration of the microbial DNA that embodiment 1 obtains is 162.8ng/ μ L, OD260/280 is that 1.98 (OD260 is the absorbance of nucleic acid, and OD280 is the absorbance of protein, when OD260/280 is between 1.8～2.0, illustrate that this DNA purity is high, it does not have be subject to the impact of protein groups and RNA)。
Comparative example
In the stomach of prior art, the extracting method of microorganism total DNA, comprises the steps:
Step 1, takes 20mg mucosa tissue, uses QIAampDNAMiniKit test kit to carry out the extraction of STb gene, obtains STb gene；
Step 2, re-uses Nanodrop2000, carries out the mensuration of DNA content；
Step 3, polymerase chain reaction (PCR) amplification: pcr amplification 16SrRNAv1-v2 district, the agarose gel electrophoresis detection that the PCR primer obtained uses mass percentage concentration to be 1%, to get rid of human genome DNA interference, it is determined that the quality of microorganism total DNA；Wherein, 16SrRNAv1-v2 primer is:
27F-5 '-GCCTTGCCAGCCCGCTCAGTCAGAGTTTGATCCTGGCTCAG-3 ' and
338R-5’GCCTCCCTCGCGCCATCAGNNNNNNNNCATGCTGCCTCCCGTAGGAGT-3’。
The concentration of the microbial DNA that comparative example obtains is 76.6ng/ μ L, OD260/280 is 1.68。
The 16SrRNAv1-v2PCR product 1% agarose gel electrophoresis figure of the DNA that Fig. 1 is embodiment 1 and comparative example obtains, as shown in Figure 1, the 16SrRNAv1-v2PCR product band of embodiment 1 is limpid in sight, the 16SrRNAv1-v2PCR product band dim of comparative example。
Sample selected by extracting method of the present invention is stomach flushing liquor, it is different from the human stomach mucosal tissue that traditional method adopts, stomach flushing liquor is practically free of human body cell and tissue, therefore the DNA overwhelming majority that stomach flushing liquor extracts as sample is adopted to come from microorganism in stomach, but not human cell tissue, thus overcome the interference of human genome DNA；Additionally adopt microorganism proprietary primer amplification 16SrRNAv1-v2 district again through PCR, carry out PCR checking, get rid of the interference of human genome DNA further。
Extracting method of the present invention, by adding equal-volume 100% ethanol, forms 50% ethanol system, secures the microorganism in stomach flushing liquor, effectively save microorganism total DNA；Removing supernatant by adding appropriate amount of buffer solution centrifugation, allowing microorganism fall in solid phase, thus eliminating the interference of gastric acid in stomach flushing liquor；Adding buffer again in solid phase, adjusting sample pH is about 7, and recentrifuge removes supernatant, removes the interference of gastric acid further, thus overcoming the interference of the low pH of sample。
Obviously, above-described embodiment is only for clearly demonstrating example of the present invention, and is not the restriction to embodiments of the present invention。For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description。Here without also cannot all of embodiment be given exhaustive。And the apparent change that these spirit belonging to the present invention are extended out or variation are still among protection scope of the present invention。
Claims (10)
1. the extracting method of microorganism total DNA in a human stomach, it is characterised in that comprise the steps:
Step 1, mixes isopyknic stomach flushing liquor and alcoholic solution, stands a period of time at-20 DEG C, by the material centrifugation at 4 DEG C after standing, removes supernatant, obtain solid phase A after centrifugation；
Step 2, adds the desired amount of buffer in the solid phase A of step 1, and mixed material carries out after mix homogeneously first time centrifugation at 4 DEG C, removes supernatant, retain solid phase after centrifugation；The solid phase of gained is carried out at 4 DEG C second time centrifugation, removes supernatant after centrifugation, obtain solid phase B；
Step 3, is sequentially added into the desired amount of E.C. 3.4.21.64 and buffer in the solid phase B of step 2, carries out heating in water bath, obtain liquid phase C after heating after mix homogeneously at 56 DEG C；
Step 4, uses DNA extraction kit to carry out the extraction of microorganism total DNA the liquid phase C of step 3, obtains STb gene, re-use micro-ultraviolet-visible spectrophotometer, carry out the mensuration of DNA total content；
Step 5, polymerase chain reaction (PCR) amplification: pcr amplification 16SrRNAv1-v2 district, the PCR primer obtained adopts agarose gel to carry out electrophoresis detection, finally gives the quality of microorganism total DNA。
2. the extracting method of microorganism total DNA in human stomach according to claim 1, it is characterized in that: in step 1, the addition volume of described stomach flushing liquor is 10～20mL, and the addition volume of described alcoholic solution is 10～20mL, wherein, the mass percentage concentration of described alcoholic solution is 100%。
3. the extracting method of microorganism total DNA in human stomach according to claim 1, it is characterised in that: in step 1, the rotating speed of described centrifugation is 15000 revs/min, and centrifugation time is 10～15 minutes。
4. the extracting method of microorganism total DNA in human stomach according to claim 1, it is characterized in that: in step 2, the formula of described buffer is: in every 1L buffer, containing 137mmol sodium chloride, 2.7mmol potassium chloride, 10mmol dipotassium hydrogen phosphate and 2mmol potassium dihydrogen phosphate。
5. the extracting method of microorganism total DNA in human stomach according to claim 1, it is characterised in that: in step 2, the addition of described buffer is 10mL。
6. the extracting method of microorganism total DNA in human stomach according to claim 1, it is characterised in that: in step 2, the rotating speed of described first time centrifugation is 15000 revs/min, and centrifugation time is 10～15 minutes；The rotating speed of described second time centrifugation is 15000 revs/min, and centrifugation time is 5 minutes。
7. the extracting method of microorganism total DNA in human stomach according to claim 1, it is characterised in that: in step 3, the addition of described E.C. 3.4.21.64 is 20 μ L, and the addition of described buffer is 200 μ L。
8. the extracting method of microorganism total DNA in human stomach according to claim 1, it is characterised in that: in step 3, the time of described heating in water bath is 60 minutes。
9. the extracting method of microorganism total DNA in human stomach according to claim 1, it is characterised in that: in step 5,16SrRNAv1-v2 primer is:
27F-5 '-GCCTTGCCAGCCCGCTCAGTCAGAGTTTGATCCTGGCTCAG-3 ' and
338R-5’GCCTCCCTCGCGCCATCAGNNNNNNNNCATGCTGCCTCCCGTAGGAGT-3’。
10. the extracting method of microorganism total DNA in human stomach according to claim 1, it is characterised in that: in step 5, the mass percentage concentration of described agarose gel is 1%。
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103834738A (en) * | 2014-03-11 | 2014-06-04 | 宁波大学 | Method for extracting and detecting IncRNA in gastric juice |
| CN104099323A (en) * | 2014-07-18 | 2014-10-15 | 西南民族大学 | DNA (Deoxyribose Nucleic Acid) extraction method for analyzing diversity of yak rumen microbe structure |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103834738A (en) * | 2014-03-11 | 2014-06-04 | 宁波大学 | Method for extracting and detecting IncRNA in gastric juice |
| CN104099323A (en) * | 2014-07-18 | 2014-10-15 | 西南民族大学 | DNA (Deoxyribose Nucleic Acid) extraction method for analyzing diversity of yak rumen microbe structure |
Non-Patent Citations (1)
| Title |
|---|
| SHIRZAD FALLAHI: ""How can I extract DNA from fish stomach contents"，https://www.researchgate.net/post", 《HTTPS://WWW.RESEARCHGATE.NET/POST》 * |
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