CN103981174B - The extracting method of microbe genome DNA in freezing primitive gut - Google Patents
The extracting method of microbe genome DNA in freezing primitive gut Download PDFInfo
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Abstract
The invention discloses the extracting method of microbe genome DNA in a kind of freezing primitive gut, combined before lysis and adopt hand breaking, the method for concussion wash-out, metal mesh filter and gradient centrifugation carries out pre-treatment to comparatively Multi-example (10g) simultaneously.The present invention was both enriched cell, solved the problem that in sample, content of microorganisms is relatively on the low side, also effectively eliminated the interference of pollutent, improve efficiency and the purity of DNA extraction.The present invention has good versatility, OD260/OD230 and OD260/OD280 of the freezing primitive gut microbe genome DNA of extraction is near the mark value, directly can carry out gene amplification and Analysis of Microbial Diversity.
Description
Technical field
The present invention relates to the extracting method of microbe genome DNA in a kind of freezing primitive gut.
Background technology
Primitive gut (intestine) is the small intestine of the healthy livestock without striking, and it produces natural casing (naturalcasings) for one of important source material of recording sausage.Putrid and deteriorated in order to prevent, primitive gut must carry out storage and transport under cold conditions, therefore the freezing primitive gut that is otherwise known as.Produce both at home and abroad at present, process the small intestine that natural casing freezing primitive gut used is mainly pig and sheep.Freezing primitive gut comes from animal intestinal, concentrate again after past excrement, cleaning and packaging in slaughterhouse and transport casing enterprise to and carry out producing, processing, therefore, freezing primitive gut pollutes in enteron aisle and extraneous microorganism is inevitable, total number of bacterial colony can reach 104 ~ 107cfu/g, many microorganisms significant in public health, in suis, Salmonellas, enterorrhagia Bacillus coil 0157: H7, sulphite reduction clostridium etc. be also included within.If produce, processing site sanitary condition be bad or storage temperature is too high, the quantity of primitive gut carrying microbe even there will be increase in processing, transportation, this is easy to threaten to Field of Animal Epidemic Disease Control and public health security, also easily in casing foreign trade, cause international dispute, affect that China's casing is produced, the sound development of processing industry.Therefore, the attention of people is caused gradually to the research of microbial diversity in freezing primitive gut.At present, the research of Bacterial diversity mostly is to the research of microbial diversity in freezing primitive gut, not only the cycle is long, workload is large, and also helpless for a large amount of uncultured microorganisms, is therefore difficult to the group's composition and the structure that reflect microorganism in freezing primitive gut truly comprehensively.
In recent years, along with the development of modern molecular biology technique particularly nucleic acid, the technology such as PCR, RELP, SSCP, DGGE widely use in environmental microorganism ecological study, and the extractive technique of microbe genome DNA is prerequisite and the basis of carrying out these work.But, domestic research and the report that there is not yet microbe genome DNA in the freezing primitive gut of extraction so far.Although have the extractive technique of animal intestinal microbe genome DNA in recent years and much study report, for the larger animal of build as northeastern tiger, panda, muroid etc., more options animal intestinal content (ight soil) is as research object; And for the less animal of body, as shrimp, fly class, mulberry borer, Dendrolimus kikuchii etc. then enteron aisle and the content thereof of adopting more together as DNA extraction sample.Abroad have at present directly to have on a small quantity and extract the report that microbe genome DNA carries out Microbial diversity Journal of Sex Research from the fresh intestinal tube of people, horse and pig, but zirconium pearl or the granulated glass sphere etc. of adopting processes sample in conjunction with grinding bead homogenizer (minibeadbeater) more, higher and the follow-up extracting method of requirement for experiment condition is loaded down with trivial details, does not utilize and promotes the use of.In addition, because in fresh animal intestinal tube and content thereof, microbe population is very big, as muroid intestinal bacteria quantity can reach 1013 ~ 1014, as long as therefore aforesaid method often processes a small amount of sample (0.2 ~ 2g) and can extract enough microbe genome DNA samples.In contrast to this, in freezing primitive gut, not only manure content is few, animal intestinal tissue content is more, and primitive gut can be decreased significantly removing bacterial number in excrement, cleaning process, therefore, aforesaid method cannot be applicable to the extraction work of microbe genome DNA in freezing primitive gut completely.
Summary of the invention
The object of the present invention is to provide the extracting method of microbe genome DNA in the freezing primitive gut that a kind of versatility is good, reliability is high, simple to operate.
Technical solution of the present invention is:
An extracting method for microbe genome DNA in freezing primitive gut, is characterized in that: comprise the following steps:
(1) the freezing primitive gut sample of normal temperature unfreezing, longitudinally cuts off primitive gut with the scissors of autoclaving process, tweezers, then shreds, stirring and evenly mixing in Bechtop;
(2) take the sample 10g after mixing and add aseptic 250mL Erlenmeyer flask, then add the sterile saline of 10mL containing 0.1%Tween80 to Erlenmeyer flask, sealing Erlenmeyer flask;
(3) Erlenmeyer flask is shaken 1min, then put 2min on ice immediately; Repeat this step 3 times;
(4) in Bechtop by 100 order metal screen filtered sample of autoclaving process, collect filtrate;
(5) casing sample in filter screen is retracted Erlenmeyer flask again, then add the sterile saline of 10mL containing 0.1%Tween80 to Erlenmeyer flask, sealing Erlenmeyer flask; Repeating step (3) ~ (4) 1 time;
(6) discard casing sample, the filtrate of twice collection is merged mixing, divide and be filled in sterile centrifugation tube, 4 DEG C, the centrifugal 5min of 400rpm;
(7) to shift in supernatant to new sterile centrifugation tube, 4 DEG C, the centrifugal 5min of 8000rpm, supernatant discarded, in precipitation, add the sterilizing ultrapure water that cumulative volume is 2mL;
(8) resuspended precipitation, 4 DEG C, the centrifugal 5min of 12000rpm, supernatant discarded, retains precipitation; Repeat this step 1 time;
(9) in precipitation, add the sterilizing ultrapure water that cumulative volume is 800 μ L, resuspended precipitation, be distributed in 2 1.5mL sterilizing Eppendorf pipes;
(10) in 2 Eppendorf pipes, isopyknic phenol, chloroform, primary isoamyl alcohol mixed solution is added with suspension respectively, and volume ratio phenol: chloroform: primary isoamyl alcohol is 25:24:1, put upside down mixing 6 ~ 10 times, 4 DEG C, the centrifugal 10min of 12000rpm, is carefully transferred in new 1.5mL sterilizing Eppendorf pipe respectively by supernatant liquor; Repeat this step 1 time;
(11) supernatant liquor that above-mentioned steps (10) obtains is added the Virahol of 0.6 times of volume, put upside down mixing ,-20 DEG C of centrifugal 10min of standing 2h, 12000rpm, supernatant discarded;
(12) add 70% ethanol 600 μ L washing precipitation, 4 DEG C, the centrifugal 5min of 12000rpm, supernatant discarded, retain precipitation, at room temperature after abundant drying, precipitate with the TE buffer solution of 20 ~ 40 μ L ,-20 DEG C save backup.
Wherein, in said extracted method, described containing 0.1% being volume ratio in 0.1%Tween80 sterile saline ().
Described phenol is the saturated phenol of domestic Tris (Tris-phenol, pH7.8 ~ 8.0), and chloroform and primary isoamyl alcohol are domestic analytical pure chemical reagent.Seasoning about 30min is in atmosphere referred to about " fully dry under room temperature ".
Described TE damping fluid is by 10mMTris-HCl (pH8.0), and 1mMEDTA (pH8.0) forms.
Advantage of the present invention and effect:
(1) the present invention be combined before lysis adopt hand breaking, the method for concussion wash-out, metal mesh filter and gradient centrifugation carries out pre-treatment to comparatively Multi-example (10g) simultaneously, both cell was enriched, solve the problem that in sample, content of microorganisms is relatively on the low side, also effectively eliminate the interference of pollutent, improve efficiency and the purity of DNA extraction.
(2) extracting method of microbe genome DNA in freezing primitive gut provided by the present invention, there is good versatility, OD260/OD230 and OD260/OD280 of freezing primitive gut microbe genome DNA extracted is near the mark value, directly can carry out gene amplification and Analysis of Microbial Diversity.
(3) requirement for experiment condition is not high, and the equipment used and reagent are laboratory conventional equipment and common agents, and general biological chemistry or microbiology laboratory all can complete, and its cost is far below import and domestic like product test kit.
(4) operation steps is simple, and the test period is short, has both saved human and material resources and financial resources, and effectively reduce to the pollution of sample and the damage of DNA molecular in operating process again, the genomic dna of extraction has higher integrity and specificity.
Accompanying drawing explanation
Below in conjunction with drawings and Examples, the invention will be further described.
Fig. 1 is microbe genome DNA agarose gel electrophoresis figure in freezing sheep primitive gut.Wherein swimming lane M is DNA molecular amount standard DL2000; Swimming lane 1-2 is that in freezing sheep primitive gut, microbe genome DNA extracts product.
Fig. 2 is the pcr amplification product agarose gel electrophoresis figure of bacterial 16 S rDNA in freezing sheep primitive gut.Wherein swimming lane M is DNA molecular amount standard DL2000; Swimming lane 1-3 is the pcr amplification product of bacterial 16 S rDNA in freezing sheep primitive gut.
Embodiment
Tween80(tween 80), Tris(tricarboxymethyl aminomethane), EDTA(ethylenediamine tetraacetic acid (EDTA)) and the saturated phenol (Tris-phenol of Tris, pH7.8 ~ 8.0) be domestic reagent, NaC, dehydrated alcohol, chloroform and primary isoamyl alcohol etc. are domestic analytical pure chemical reagent, PCR kit (16SrDNABacterialIdentificationPCRKit) is precious biotechnology (Dalian) Products (article No.: D310), and metal screen is Dao Xu Zhang Xingshashai factory of Shangyu city, Zhejiang product.
(1) take out sealing, freezing sheep primitive gut sample from refrigerator, thaw under putting normal temperature, first primitive gut is longitudinally cut off with the scissors of autoclaving process, tweezers in Bechtop and fully shred again, with tweezers stirring and evenly mixing repeatedly.
(2) take the sample 10g after mixing and add aseptic 250mL Erlenmeyer flask, then add 10mL sterile saline (containing 0.1%Tween80) to Erlenmeyer flask, sealing Erlenmeyer flask.
(3) by Erlenmeyer flask whirlpool concussion 1min, 2min is on ice put immediately.Repeat this step 3 times.
(4) in Bechtop by 100 order metal screen filtered sample of autoclaving process, collect filtrate.
(5) casing sample in filter screen is retracted Erlenmeyer flask again, then add 10mL sterile saline (containing 0.1%Tween80) to Erlenmeyer flask, sealing Erlenmeyer flask.Repeating step (3) ~ (4) 1 time.
(6) discard casing sample, the filtrate of twice collection is merged mixing, divide and be filled in sterile centrifugation tube, 4 DEG C, the centrifugal 5min of 400rpm.
(7) to shift in supernatant to new sterile centrifugation tube, 4 DEG C, the centrifugal 5min of 8000rpm, supernatant discarded, in precipitation, add the sterilizing ultrapure water that cumulative volume is 2mL.
(8) resuspended precipitation, 4 DEG C, the centrifugal 5min of 12000rpm, supernatant discarded, retains precipitation.Repeat this step 1 time.
(9) in precipitation, add the sterilizing ultrapure water that cumulative volume is 800 μ L, resuspended precipitation, be distributed in 2 1.5mL sterilizing Eppendorf pipes.
(10) in 2 Eppendorf pipes, isopyknic phenol is added with suspension respectively: chloroform: primary isoamyl alcohol (25:24:1), put upside down mixing 6 ~ 10 times, 4 DEG C, the centrifugal 10min of 12000rpm, is carefully transferred to supernatant liquor in new 1.5mL sterilizing Eppendorf pipe respectively.Repeat this step 1 time.
(11) supernatant liquor that above-mentioned steps (10) obtains is added the Virahol of 0.6 times of volume, put upside down mixing ,-20 DEG C of centrifugal 10min of standing 2h, 12000rpm, supernatant discarded.
(12) add 70% ethanol 600 μ L washing precipitation, 4 DEG C, the centrifugal 5min of 12000rpm, supernatant discarded, retain precipitation, under room temperature after abundant drying, precipitate with the TE buffer solution of 20 ~ 40 μ L ,-20 DEG C save backup (participation Fig. 1).
With spectrophotometric determination OD230, OD260, OD280, result shows OD260/OD230=2.04, OD260/OD280=1.83 is near the mark value (OD260/OD230 standard value is 2.0, OD260/OD280 standard value is 1.8), can directly apply to microbial gene amplification and diversity analysis.
With the genomic dna of said extracted for template, with precious biotechnology (Dalian) Products of 16SrDNABacterialIdentificationPCRKit(, article No.: D310) increase bacterial 16 S rDNA, PCR reaction system and condition are carried out see test kit specification sheets, and PCR primer 1.0% sepharose carries out electrophoresis (participation Fig. 2).
As can be seen from the above results, the inventive method is not only simple to operate, with low cost, and high-quality DNA can be obtained, be that template carries out PCR with the microbe genome DNA that present method is extracted, can Successful amplification with estimate the product of about 1700bp of the same size, band brightness is strong, specificity is good, can directly apply to Analysis of Microbial Diversity.
Finally it is pointed out that specific embodiment described herein only in order to technical scheme of the present invention to be described, be not intended to limit the present invention.In addition should be understood that those skilled in the art can modify to the technical scheme of invention or equivalent replacement, and do not depart from the spirit and scope of technical solution of the present invention, it all should be encompassed in right of the present invention.
Claims (1)
1. the extracting method of microbe genome DNA in freezing primitive gut, is characterized in that: comprise the following steps:
(1) the freezing primitive gut sample of normal temperature unfreezing, longitudinally cuts off primitive gut with the scissors of autoclaving process, tweezers, then shreds, stirring and evenly mixing in Bechtop;
(2) take the sample 10g after mixing and add aseptic 250mL Erlenmeyer flask, then add the sterile saline of 10mL containing 0.1%Tween80 to Erlenmeyer flask, sealing Erlenmeyer flask;
(3) Erlenmeyer flask is shaken 1min, then put 2min on ice immediately; Repeat this step 3 times;
(4) in Bechtop by 100 order metal screen filtered sample of autoclaving process, collect filtrate;
(5) casing sample in filter screen is retracted Erlenmeyer flask again, then add the sterile saline of 10mL containing 0.1%Tween80 to Erlenmeyer flask, sealing Erlenmeyer flask; Repeating step (3) ~ (4) 1 time;
(6) discard casing sample, the filtrate of twice collection is merged mixing, divide and be filled in sterile centrifugation tube, 4 DEG C, the centrifugal 5min of 400rpm;
(7) to shift in supernatant to new sterile centrifugation tube, 4 DEG C, the centrifugal 5min of 8000rpm, supernatant discarded, in precipitation, add the sterilizing ultrapure water that cumulative volume is 2mL;
(8) resuspended precipitation, 4 DEG C, the centrifugal 5min of 12000rpm, supernatant discarded, retains precipitation; Repeat this step 1 time;
(9) in precipitation, add the sterilizing ultrapure water that cumulative volume is 800 μ L, resuspended precipitation, be distributed in 2 1.5mL sterilizing Eppendorf pipes;
(10) in 2 Eppendorf pipes, isopyknic phenol, chloroform, primary isoamyl alcohol mixed solution is added with suspension respectively, and volume ratio phenol: chloroform: primary isoamyl alcohol is 25:24:1, put upside down mixing 6 ~ 10 times, 4 DEG C, the centrifugal 10min of 12000rpm, is carefully transferred in new 1.5mL sterilizing Eppendorf pipe respectively by supernatant liquor; Repeat this step 1 time;
(11) supernatant liquor that above-mentioned steps (10) obtains is added the Virahol of 0.6 times of volume, put upside down mixing ,-20 DEG C of centrifugal 10min of standing 2h, 12000rpm, supernatant discarded;
(12) add 70% ethanol 600 μ L washing precipitation, 4 DEG C, the centrifugal 5min of 12000rpm, supernatant discarded, retain precipitation, at room temperature after abundant drying, precipitate with the TE buffer solution of 20 ~ 40 μ L ,-20 DEG C save backup.
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| CN102978198A (en) * | 2012-11-29 | 2013-03-20 | 上海市农业科学院 | Microbial genome DNA (deoxyribonucleic acid) indirect extraction method for evaluating diversity of animal intestinal microflora |
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| 65株冷冻肠衣分离细菌的鉴定;仇保丰;《中国食品卫生杂志》;20131231;第25卷(第5期);材料和方法部分 * |
| 史氏鲟肠道微生物基因组DNA提取方法的研究;张旭;《湖南农业科学》;20111231;全文 * |
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