CN105695451B - The extracting method of microorganism total DNA in a kind of human stomach - Google Patents
The extracting method of microorganism total DNA in a kind of human stomach Download PDFInfo
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- CN105695451B CN105695451B CN201610243307.3A CN201610243307A CN105695451B CN 105695451 B CN105695451 B CN 105695451B CN 201610243307 A CN201610243307 A CN 201610243307A CN 105695451 B CN105695451 B CN 105695451B
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- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
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Abstract
The invention discloses a kind of extracting methods of microorganism total DNA in human stomach, extracting method of the present invention is the pre-treatment step that joined on the basis of existing extracting method to stomach extracting solution, to efficiently solve the problems, such as that extraction efficiency caused by interfering due to the low pH factor of sample and human genome DNA is low and effect is poor, extracting method of the invention is more than doubled compared to its extraction efficiency of existing method, extracted microbial DNA concentration is high, microorganism total DNA occupation ratio is high, the area 16S rRNA v1-v2 PCR product band is limpid in sight, and then the microbial status in stomach can be more accurately reacted comprehensively, research for subsequent biological community structure and function is laid a good foundation.
Description
Technical field
The present invention relates to a kind of extracting methods of microorganism total DNA in human stomach, belong to biological technology field.
Background technique
Stomach is the pith of digestion, since the microorganism that the secretion of gastric acid makes it be included becomes alimentary canal
Special a part in microbial ecosystem, the health and disease of complicated microbiologic population and its metabolite and human body in stomach
Sick close relation, human energy metabolism, nutrient absorption, immune system, in terms of play important work
With.Under one's belt in the research process of the structure of community of microorganism, the extraction of microorganism total DNA is particularly important.However due to
Bacterial abundance is lower in stomach and the particular surroundings of low pH, causes microorganism total DNA extraction efficiency in stomach low, and extract
Effect is poor.Common extracting mode, as experiment sample, cooperates microbial DNA extracts kit to be mentioned using mucosa tissue
It takes, inefficiency, and most of human genome DNA for mucosa tissue, few part is the DNA of microorganism.In this way
Extract resulting DNA carry out subsequent molecular ecology research cannot biological community structure and function in comprehensive and accurate reaction stomach
It can situation.Therefore a kind of stomach microbial total that can effectively solve the problem that the low pH factor of sample and human genome DNA and bring interference
The exploitation of DNA extraction method is necessary.
Summary of the invention
Goal of the invention: technical problem to be solved by the invention is to provide a kind of extractions of microorganism total DNA in human stomach
Method, this method can greatly improve the Genome DNA extraction efficiency and quality of microorganism in stomach.
In order to solve the above technical problems, the technical scheme adopted by the invention is as follows:
The extracting method of microorganism total DNA, includes the following steps: in a kind of human stomach
Step 1, isometric stomach flushing liquor and ethanol solution are mixed, a period of time is stood at -20 DEG C, after standing
Material be centrifugated at 4 DEG C, supernatant is removed after centrifuge separation, obtains solid phase A；
Step 2, the desired amount of buffer is added into the solid phase A of step 1, after mixing by mixed material at 4 DEG C
First time centrifuge separation is carried out, supernatant is removed after centrifuge separation, retains solid phase；Resulting solid phase is carried out second at 4 DEG C
Secondary centrifuge separation removes supernatant after centrifuge separation, obtains solid phase B；
Step 3, the desired amount of Proteinase K and buffer are sequentially added into the solid phase B of step 2, after mixing in 56
Heating water bath is carried out at DEG C, and liquid phase C is obtained after heating；
Step 4, the extraction that the liquid phase C of step 3 is carried out to total DNA using DNA extraction kit, obtains total DNA, reuses
Micro-ultraviolet-visible spectrophotometer carries out the measurement of DNA total content；
Step 5, polymerase chain reaction amplification: the area PCR amplification 16S rRNAv1-v2, obtained PCR product use agar
Sugared gel carries out electrophoresis detection, finally obtains the quality of microorganism total DNA.
Wherein, in step 1, the addition volume of the stomach flushing liquor is 10~20mL, the addition volume of the ethanol solution
For 10~20mL, wherein the mass percentage concentration of the ethanol solution is 100%.
Wherein, in step 1, the revolving speed of the centrifuge separation is 15000 revs/min, and centrifugation time is 10~15 minutes.
Wherein, in step 2, the buffer is grouped as by the group 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 additional amount of the buffer is 10mL.
Wherein, in step 2, the revolving speed of the first time centrifuge separation is 15000 revs/min, and centrifugation time is 10~15
Minute；The revolving speed of second of centrifuge separation is 15000 revs/min, and centrifugation time is 5 minutes.
Wherein, in step 3, the additional amount of the Proteinase K is 20 μ L, and the additional amount of the buffer is 200 μ L.
Wherein, in step 3, the time of the heating water bath is 60 minutes.
Wherein, in step 5,16S rRNAv1-v2 primer are as follows:
27F-5 '-GCCTTGCCAGCCCGCTCAGTCAGAGTTTGATCCTGGCTCAG-3 ' and
338R-5’GCCTCCCTCGCGCCATCAGNNNNNNNNCATGCTGCCTCCCGTAGGAGT-3’。
Wherein, in step 5, the mass percentage concentration of the Ago-Gel is 1%.
The utility model has the advantages that extracting DNA method compared to existing gastric mucosa, the present invention extracts the side of microorganism total DNA in stomach
Method is efficiently solved interfered due to the low pH factor of sample and human genome DNA caused by extraction efficiency is low and effect difference is asked
Topic, extraction efficiency are more than doubled, and extracted microbial DNA concentration is high, and microorganism total DNA occupation ratio is high, 16S
The area rRNAv1-v2 PCR product band is limpid in sight, and then can more accurately react the microbial status in stomach comprehensively, after being
The research of continuous biological community structure and function is laid a good foundation.
Detailed description of the invention
Fig. 1 is the PCR product band contrast effect figure that the embodiment of the present invention 1 and comparative example obtain.
Specific embodiment
Below in conjunction with attached drawing, technical scheme is described further, but the scope of protection of present invention is simultaneously
It is not limited to this.
Embodiment 1
The extracting method of microorganism total DNA, specifically comprises the following steps: in stomach of the present invention
Step 1, the ethanol solution that stomach flushing liquor obtained in 15mL clinic and 15mL mass percentage concentration are 100% is mixed
It closes, saves for 24 hours, the material after preservation is placed in constant speed centrifuge at 4 DEG C, using 15000 revs/min at -20 DEG C
Revolving speed is centrifuged 10min, removes supernatant after centrifugation, obtains solid phase A；
Step 2,10mL buffer is added into solid phase A (if the additional amount of amount of buffer is too low, cannot completely eliminate stomach
The brought low pH interference of acid, if the additional amount of amount of buffer is too high, has more microorganism to remain in liquid phase and lost
Abandon, influence the extraction efficiency of DNA), it is uniformly mixed using oscillator oscillation 2min, after mixing by mixed material at 4 DEG C
It is placed in constant speed centrifuge, is centrifuged 10min using 15000 revs/min of revolving speeds, supernatant is removed after centrifuge separation, retain solid
Phase；Resulting solid phase is continued to be placed in constant speed centrifuge at 4 DEG C, is centrifuged 5min using 15000 revs/min of revolving speeds, from
Supernatant is removed after heart separation, obtains solid phase B；The supernatant of centrifugation removal twice, further eliminates gastric acid in stomach flushing liquor
Interference；
Step 3,20 μ L Proteinase Ks and 200 μ L buffers are sequentially added into solid phase B, it is mixed using oscillator oscillation 2min
It closes uniformly, is uniformly mixed to be placed in 56 DEG C of water-baths and heat 60 minutes, guarantee that Proteinase K sufficiently reacts, obtain liquid phase C；
Step 4, the extraction that liquid phase C is carried out to microorganism total DNA using QIAamp DNA Mini Kit kit, obtains
Total DNA；Nanodrop 2000 is reused, the measurement of DNA content is carried out.
Step 5, polymerase chain reaction amplification: the area PCR amplification 16S rRNA v1-v2, obtained PCR product use matter
It measures the agarose gel electrophoresis that percentage concentration is 1% to detect, to exclude human genome DNA interference, determines microorganism total DNA
Quality；Wherein, 16S rRNAv1-v2 primer are as follows:
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, and gained solid phase sample is DNA after pretreatment
The best applied sample amount of extracts kit, stomach flushing liquor are lower than 10mL, then mentioned DNA concentration is lower, do not meet follow-up test demand, high
In 20mL, then DNA concentration will not increase accordingly, and increase the influence of gastric acid instead.
The formula of buffer used in extracting method of the present invention are as follows: in every 1L buffer, containing 137mmol sodium chloride,
2.7mmol potassium chloride, 10mmol dipotassium hydrogen phosphate and 2mmol potassium dihydrogen phosphate.It, will using concentrated hydrochloric acid when using the buffer
The pH value adjustment of buffer is to 7.4.Suitable buffer is added in the sample, the influence of gastric acid in sample can be overcome, and
Make the pH 7 or so of system.
The concentration for the microbial DNA that embodiment 1 obtains is 162.8ng/ μ L, and OD260/280 is that 1.98 (OD260 is nucleic acid
Absorbance, OD280 is the absorbance of protein, when OD260/280 is between 1.8~2.0, illustrates DNA purity is high, does not have
It is influenced by protein groups and RNA).
Comparative example
The extracting method of microorganism total DNA, includes the following steps: in the stomach of the prior art
Step 1,20mg mucosa tissue is taken, the extraction of total DNA is carried out using QIAamp DNA Mini Kit kit,
Obtain total DNA；
Step 2, Nanodrop 2000 is reused, the measurement of DNA content is carried out；
Step 3, polymerase chain reaction amplification: the area PCR amplification 16S rRNA v1-v2, obtained PCR product use matter
It measures the agarose gel electrophoresis that percentage concentration is 1% to detect, to exclude human genome DNA interference, determines microorganism total DNA
Quality；Wherein, 16S rRNAv1-v2 primer are as follows:
27F-5 '-GCCTTGCCAGCCCGCTCAGTCAGAGTTTGATCCTGGCTCAG-3 ' and
338R-5’GCCTCCCTCGCGCCATCAGNNNNNNNNCATGCTGCCTCCCGTAGGAGT-3’。
The concentration for the microbial DNA that comparative example obtains is 76.6ng/ μ L, OD260/280 1.68.
Fig. 1 is 1% Ago-Gel of 16S rRNAv1-v2PCR product for the DNA that embodiment 1 and comparative example obtain
Electrophoretogram, as shown in Figure 1, the 16S rRNAv1-v2PCR product band of embodiment 1 is limpid in sight, the 16S of comparative example
RRNAv1-v2PCR product band dim and blur.
Sample selected by extracting method of the present invention is stomach flushing liquor, is different from human body gastric mucosa group used by conventional method
It knits, stomach flushing liquor is practically free of human body cell and tissue, therefore big absolutely using the DNA that stomach flushing liquor is extracted as sample
Part microorganism in stomach, rather than human cell tissue, to overcome the interference of human genome DNA；In addition lead to again
PCR is crossed using the proprietary area primer amplification 16S rRNAv1-v2 of microorganism, PCR verifying is carried out, further excludes human genome
The interference of DNA.
Extracting method of the present invention forms 50% alcohol system, secures stomach flushing liquor by the way that isometric 100% ethyl alcohol is added
In microorganism, effectively save microorganism total DNA；By the way that appropriate amount of buffer solution is added and is centrifugated removal supernatant, allow
Microorganism falls in solid phase, to eliminate the interference of gastric acid in stomach flushing liquor；Buffer is added into solid phase again, adjusts sample
This pH is 7 or so, and centrifugation removal supernatant, further removes the interference of gastric acid, to overcome the interference of the low pH of sample again.
Obviously, the above embodiment is merely an example for clearly illustrating the present invention, and is not to of the invention
The restriction of embodiment.For those of ordinary skill in the art, it can also be made on the basis of the above description
Its various forms of variation or variation.There is no necessity and possibility to exhaust all the enbodiments.And these belong to this hair
The obvious changes or variations that bright spirit is extended out are still in the protection scope of this invention.
Claims (9)
1. the extracting method of microorganism total DNA in a kind of human stomach, which comprises the steps of:
Step 1, isometric stomach flushing liquor and ethanol solution are mixed, a period of time is stood at -20 DEG C, by the object after standing
Expect to be centrifugated at 4 DEG C, supernatant is removed after centrifuge separation, obtains solid phase A；
Step 2, the desired amount of buffer is added into the solid phase A of step 1, after mixing carries out mixed material at 4 DEG C
It is centrifugated for the first time, supernatant is removed after centrifuge separation, retain solid phase；Resulting solid phase is carried out at 4 DEG C second from
Heart separation, removes supernatant after centrifuge separation, obtains solid phase B；The formula of the buffer are as follows: in every 1L buffer, contain
137mmol sodium chloride, 2.7mmol potassium chloride, 10mmol dipotassium hydrogen phosphate and 2mmol potassium dihydrogen phosphate；
Step 3, the desired amount of Proteinase K and buffer are sequentially added into the solid phase B of step 2, after mixing at 56 DEG C
Heating water bath is carried out, liquid phase C is obtained after heating；
Step 4, the extraction that the liquid phase C of step 3 is carried out to microorganism total DNA using DNA extraction kit, obtains total DNA, then make
With micro-ultraviolet-visible spectrophotometer, the measurement of DNA total content is carried out；
Step 5, polymerase chain reaction amplification: the area PCR amplification 16S rRNA v1-v2, obtained PCR product use agarose
Gel carries out electrophoresis detection, finally obtains the quality of microorganism total DNA.
2. the extracting method of microorganism total DNA in human stomach according to claim 1, it is characterised in that: in step 1, institute
The addition volume for stating stomach flushing liquor is 10~20mL, and the addition volume of the ethanol solution is 10~20mL, wherein the ethyl alcohol
The mass percentage concentration of 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, institute
The revolving speed for stating centrifuge separation 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 characterised in that: in step 2, institute
The additional amount for stating buffer is 10mL.
5. the extracting method of microorganism total DNA in human stomach according to claim 1, it is characterised in that: in step 2, institute
The revolving speed for stating centrifuge separation for the first time is 15000 revs/min, and centrifugation time is 10~15 minutes；Second of centrifuge separation
Revolving speed be 15000 revs/min, centrifugation time be 5 minutes.
6. the extracting method of microorganism total DNA in human stomach according to claim 1, it is characterised in that: in step 3, institute
The additional amount for stating Proteinase K is 20 μ L, and the additional amount of the buffer is 200 μ L.
7. the extracting method of microorganism total DNA in human stomach according to claim 1, it is characterised in that: in step 3, institute
The time for stating heating water bath is 60 minutes.
8. the extracting method of microorganism total DNA in human stomach according to claim 1, it is characterised in that: in step 5,16S
RRNA v1-v2 primer are as follows:
27F-5 '-GCCTTGCCAGCCCGCTCAGTCAGAGTTTGATCCTGGCTCAG-3 ' and
338R-5’GCCTCCCTCGCGCCATCAGNNNNNNNNCATGCTGCCTCCCGTAGGAGT-3’。
9. the extracting method of microorganism total DNA in human stomach according to claim 1, it is characterised in that: in step 5, institute
The mass percentage concentration for stating Ago-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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2016
- 2016-04-19 CN CN201610243307.3A patent/CN105695451B/en active Active
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 |
|---|
| "How can I extract DNA from fish stomach contents"，https://www.researchgate.net/post;Shirzad Fallahi;《https://www.researchgate.net/post》;20141231;第1页 |
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