CN103981174A - Method for extracting microbial genome DNA (Deoxyribose Nucleic Acid) of frozen archenteron - Google Patents

Abstract

The invention discloses a method for extracting microbial genome DNA (Deoxyribose Nucleic Acid) of frozen archenteron. According to the method, a relatively large amount of samples (10g) are subjected to pretreatment by using a method with the combination of artificial crushing, eluting based on oscillation, filtration based on a metal net and gradient centrifugation before cell lysis. By adopting the method, cells are enriched, the problem that the content of microorganisms in the samples is relatively low is solved, the interference of pollutants is effectively prevented, and the DNA extraction efficiency and the DNA extraction purity are improved. The method is relatively good in university, the OD260/OD230 and OD/260/OD280 of the microbial genome DNA of the microbial genome are approximate to standard values, and thus the DNA can be directly subjected to gene amplification and microbial diversity analysis.

Description

The extracting method of microbe genome DNA in freezing primitive gut

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 without the healthy livestock of striking, and it is to produce natural casing (natural casings) and for recording one of important source material of sausage.Putrid and deteriorated in order to prevent, primitive gut must carry out storage and transport under freezing condition, therefore the freezing primitive gut that is otherwise known as.The small intestine produce both at home and abroad at present, processing natural casing freezing primitive gut used being mainly pig and sheep.Freezing primitive gut comes from animal intestinal, in slaughterhouse, after past excrement, cleaning and packing, concentrate again and transport casing enterprise to and produce, process, therefore, freezing primitive gut pollutes in enteron aisle and extraneous microorganism is inevitable, total number of bacterial colony can reach 104～107 cfu/g, many microorganisms significant aspect public health, as in suis, Salmonellas, enterorrhagia Bacillus coil 0157: H7, sulphite reduction clostridium etc. is also included within.If produced, processing site sanitary condition is 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 the sound development of China's casing production, processing industry.Therefore, the research of microbial diversity in freezing primitive gut is caused to people's attention gradually.At present, to the research of microbial diversity in freezing primitive gut mostly be can culturing micro-organisms research, not only the cycle is long, workload is large, and in a large number can not culturing micro-organisms also helpless, be therefore difficult to reflect truly that the group of microorganism in freezing primitive gut forms and structure comprehensively.

In recent years, along with the particularly development of nucleic acid of modern molecular biology technique, the technology such as PCR, RELP, SSCP, DGGE have been widely used 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 extracts microbe genome DNA in freezing primitive gut that there is not yet so far.Although in recent years the extractive technique of animal intestinal microbe genome DNA is had to a lot of research 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. adopt enteron aisles and contents thereof together as DNA extraction sample more.External having on a small quantity directly has from the fresh intestinal tube of people, horse and pig and extracts the report that microbe genome DNA carries out Microbial diversity Journal of Sex Research at present, but zirconium pearl or the granulated glass spherees etc. of adopting are processed sample in conjunction with grinding bead homogenizer (minibead beater) 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 microbe population in fresh animal intestinal tube and content thereof is very big, as muroid intestinal bacteria quantity can reach 1013～1014, so aforesaid method often can extract enough microbe genome DNA samples as long as process a small amount of sample (0.2～2 g).In contrast to this, in freezing primitive gut, not only ight soil content is few, animal intestinal tissue content is more, and primitive gut bacterial number in going excrement, cleaning process can be decreased significantly, 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, scissors, the tweezers in Bechtop, with autoclaving, processed are longitudinally cut off primitive gut, then shred stirring and evenly mixing;

(2) sample 10 g that take after mixing add 250 aseptic mL Erlenmeyer flasks, then add 10 mL containing the sterile saline of 0.1% Tween80 to Erlenmeyer flask, sealing Erlenmeyer flask;

(3) Erlenmeyer flask is shaken to 1 min, then put immediately 2 min on ice; Repeat this step 3 times;

(4) the 100 order metal screen filtered sample of processing with autoclaving in Bechtop, collect filtrate;

(5) casing sample in filter screen is retracted to Erlenmeyer flask again, then add 10 mL containing the sterile saline of 0.1% Tween80 to Erlenmeyer flask, sealing Erlenmeyer flask; Repeating step (3)～(4) 1 time;

(6) discard casing sample, the merging of the filtrate of twice collection is mixed, divide and be filled in sterilizing centrifuge tube, 4 ℃, centrifugal 5 min of 400 rpm;

(7) shift supernatant to new sterilizing centrifuge tube, 4 ℃, centrifugal 5 min of 8000 rpm, supernatant discarded, in precipitation and to add cumulative volume be the sterilizing ultrapure water of 2 mL;

(8) resuspended precipitation, 4 ℃, centrifugal 5 min of 12 000 rpm, supernatant discarded, retains precipitation; Repeat this step 1 time;

(9) in precipitation, adding cumulative volume is the sterilizing ultrapure water of 800 μ L, and resuspended precipitation is distributed in 2 1.5 mL sterilizing Eppendorf pipes;

(10) in 2 Eppendorf pipes, add respectively and the isopyknic phenol of suspension, chloroform, primary isoamyl alcohol mixed solution, and volume ratio phenol: chloroform: primary isoamyl alcohol is 25:24:1, put upside down and mix 6～10 times, 4 ℃, centrifugal 10 min of 12 000 rpm, are carefully transferred to supernatant liquor respectively in 1.5 new mL sterilizing Eppendorf pipes; Repeat this step 1 time;

(11) supernatant liquor above-mentioned steps (10) being obtained adds the Virahol of 0.6 times of volume, puts upside down and mixes ,-20 ℃ of standing 2 h, centrifugal 10 min of 12 000 rpm, supernatant discarded;

(12) add 70% ethanol 600 μ L washing precipitations, 4 ℃, centrifugal 5 min of 12 000 rpm, supernatant discarded, retains precipitation, and after being at room temperature fully dried, with the TE damping fluid dissolution precipitation of 20～40 μ L ,-20 ℃ save backup.

Wherein, in said extracted method, describedly contain in 0.1% Tween80 sterile saline () 0.1% for volume ratio.

Described phenol is the saturated phenol of domestic Tris (Tris-phenol, pH 7.8～8.0), and chloroform and primary isoamyl alcohol are domestic analytical pure chemical reagent.About " fully dry under room temperature ", refer to seasoning 30 min left and right in air.

Described TE damping fluid is by 10 mM Tris-HCl (pH 8.0), and 1 mM EDTA (pH 8.0) forms.

Advantage of the present invention and effect:

(1) the present invention is in conjunction with adopting the method for hand breaking, concussion wash-out, metal mesh filter and gradient centrifugation simultaneously to carrying out pre-treatment compared with Multi-example (10 g) before lysis, both enrichment cell, solved content of microorganisms problem relatively on the low side in sample, also effectively remove the interference of pollutent, improved 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 the OD260/OD280 value of being near the mark of the freezing primitive gut microbe genome DNA extracting, can directly carry out gene amplification and Analysis of Microbial Diversity.

(3) requirement for experiment condition is not high, and the equipment using 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, effectively reduces again the damage to the pollution of sample and DNA molecular in operating process, and 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 DL2 000; 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 DL2 000; Swimming lane 1-3 is the pcr amplification product of bacterial 16 S rDNA in freezing sheep primitive gut.

Embodiment

Tris(tricarboxylic ylmethyl aminomethane), EDTA(ethylenediamine tetraacetic acid (EDTA)) and the saturated phenol (Tris-phenol of Tris Tween80(tween 80),, pH 7.8～8.0) be domestic reagent, NaC, dehydrated alcohol, chloroform and primary isoamyl alcohol etc. are domestic analytical pure chemical reagent, PCR test kit (16S rDNA Bacterial Identification PCR Kit) is precious biotechnology (Dalian) company product (article No.: D310), metal screen is Shangyu city, Zhejiang Dao Xu Zhang Xingshashai factory product.

(1) from refrigerator, take out sealing, freezing sheep primitive gut sample, put under normal temperature and thaw, scissors, the tweezers in Bechtop, with autoclaving, processed are first longitudinally cut off primitive gut again and are fully shredded, with tweezers stirring and evenly mixing repeatedly.

(2) sample 10 g that take after mixing add 250 aseptic mL Erlenmeyer flasks, then add 10 mL sterile salines (containing 0.1% Tween80), sealing Erlenmeyer flask to Erlenmeyer flask.

(3) Erlenmeyer flask whirlpool is shaken to 1 min, put immediately 2 min on ice.Repeat this step 3 times.

(4) the 100 order metal screen filtered sample of processing with autoclaving in Bechtop, collect filtrate.

(5) casing sample in filter screen is retracted to Erlenmeyer flask again, then add 10 mL sterile salines (containing 0.1% Tween80), sealing Erlenmeyer flask to Erlenmeyer flask.Repeating step (3)～(4) 1 time.

(6) discard casing sample, the merging of the filtrate of twice collection is mixed, divide and be filled in sterilizing centrifuge tube, 4 ℃, centrifugal 5 min of 400 rpm.

(7) shift supernatant to new sterilizing centrifuge tube, 4 ℃, centrifugal 5 min of 8000 rpm, supernatant discarded, in precipitation and to add cumulative volume be the sterilizing ultrapure water of 2 mL.

(8) resuspended precipitation, 4 ℃, centrifugal 5 min of 12 000 rpm, supernatant discarded, retains precipitation.Repeat this step 1 time.

(9) in precipitation, adding cumulative volume is the sterilizing ultrapure water of 800 μ L, and resuspended precipitation is distributed in 2 1.5 mL sterilizing Eppendorf pipes.

(10) in 2 Eppendorf pipes, add respectively and the isopyknic phenol of suspension: chloroform: primary isoamyl alcohol (25:24:1), put upside down and mix 6～10 times, 4 ℃, centrifugal 10 min of 12 000 rpm, are carefully transferred to supernatant liquor respectively in 1.5 new mL sterilizing Eppendorf pipes.Repeat this step 1 time.

(11) supernatant liquor above-mentioned steps (10) being obtained adds the Virahol of 0.6 times of volume, puts upside down and mixes ,-20 ℃ of standing 2 h, centrifugal 10 min of 12 000 rpm, supernatant discarded.

(12) add 70% ethanol 600 μ L washing precipitations, 4 ℃, centrifugal 5 min of 12 000 rpm, supernatant discarded, retains precipitation, and after being fully dried under room temperature, with the TE damping fluid dissolution precipitation of 20～40 μ L ,-20 ℃ save backup (participation Fig. 1).

With spectrophotometric determination OD230, OD260, OD280, result shows OD260/OD230=2.04, OD260/OD280=1.83 value of being near the mark (OD260/OD230 standard value is that 2.0, OD260/OD280 standard value is 1.8), can directly apply to microbial gene amplification and diversity analysis.

The genomic dna of said extracted of take is template, with the precious biotechnology (Dalian) of 16S rDNA Bacterial Identification PCR Kit(company product, article No.: D310) amplification bacterial 16 S rDNA, PCR reaction system and condition are carried out referring to test kit specification sheets, and PCR product carries out electrophoresis (participation Fig. 2) with 1.0% sepharose.

From the above results, can find out, the inventive method is not only simple to operate, with low cost, and can obtain high-quality DNA, with the microbe genome DNA that present method is extracted, be that template is carried out PCR, can successfully increase and the product of estimating approximately 1 700 bp 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, is not intended to limit the present invention.In addition should be understood that those skilled in the art can modify or be equal to replacement the technical scheme of invention, and do not depart from the spirit and scope of technical solution of the present invention, it all should be encompassed in claim scope of the present invention.

Claims (1)

1. 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, scissors, the tweezers in Bechtop, with autoclaving, processed are longitudinally cut off primitive gut, then shred stirring and evenly mixing;

(2) sample 10 g that take after mixing add 250 aseptic mL Erlenmeyer flasks, then add 10 mL containing the sterile saline of 0.1% Tween80 to Erlenmeyer flask, sealing Erlenmeyer flask;

(3) Erlenmeyer flask is shaken to 1 min, then put immediately 2 min on ice; Repeat this step 3 times;

(4) the 100 order metal screen filtered sample of processing with autoclaving in Bechtop, collect filtrate;

(5) casing sample in filter screen is retracted to Erlenmeyer flask again, then add 10 mL containing the sterile saline of 0.1% Tween80 to Erlenmeyer flask, sealing Erlenmeyer flask; Repeating step (3)～(4) 1 time;

(6) discard casing sample, the merging of the filtrate of twice collection is mixed, divide and be filled in sterilizing centrifuge tube, 4 ℃, centrifugal 5 min of 400 rpm;

(7) shift supernatant to new sterilizing centrifuge tube, 4 ℃, centrifugal 5 min of 8000 rpm, supernatant discarded, in precipitation and to add cumulative volume be the sterilizing ultrapure water of 2 mL;

(8) resuspended precipitation, 4 ℃, centrifugal 5 min of 12 000 rpm, supernatant discarded, retains precipitation; Repeat this step 1 time;

(9) in precipitation, adding cumulative volume is the sterilizing ultrapure water of 800 μ L, and resuspended precipitation is distributed in 2 1.5 mL sterilizing Eppendorf pipes;

(10) in 2 Eppendorf pipes, add respectively and the isopyknic phenol of suspension, chloroform, primary isoamyl alcohol mixed solution, and volume ratio phenol: chloroform: primary isoamyl alcohol is 25:24:1, put upside down and mix 6～10 times, 4 ℃, centrifugal 10 min of 12 000 rpm, are carefully transferred to supernatant liquor respectively in 1.5 new mL sterilizing Eppendorf pipes; Repeat this step 1 time;

(11) supernatant liquor above-mentioned steps (10) being obtained adds the Virahol of 0.6 times of volume, puts upside down and mixes ,-20 ℃ of standing 2 h, centrifugal 10 min of 12 000 rpm, supernatant discarded;

(12) add 70% ethanol 600 μ L washing precipitations, 4 ℃, centrifugal 5 min of 12 000 rpm, supernatant discarded, retains precipitation, and after being at room temperature fully dried, with the TE damping fluid dissolution precipitation of 20～40 μ L ,-20 ℃ save backup.