CN103952468A - DGGE analysis method for bacterial diversity in northeast naturally-fermented sauerkraut - Google Patents
DGGE analysis method for bacterial diversity in northeast naturally-fermented sauerkraut Download PDFInfo
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Abstract
The invention relates to a DGGE analysis method for bacterial diversity in northeast naturally-fermented sauerkraut, wherein problem that the traditional method can not completely and accurately reflect the population structure and the bacterial diversity in sauerkraut is mainly solved. The method comprises: 1, pre-treating a sauerkraut sample; 2, extracting the total DNA of microorganism in the sauerkraut sample; 3, adopting the extracted total DNA of the microorganism as a template, and carrying out 16SrRNA PCR amplification; and 4, carrying out DGGE electrophoresis analysis on the PCR product, carrying out staining and imaging with a scanner after completing the electrophoresis, labeling the specific band on the PCR-DGGE spectrum, cutting the gel, recovering, and carrying out Blast comparison in the NCBI database to obtain the microorganism information so as to complete the DGGE analysis method for bacterial diversity in the northeast naturally-fermented sauerkraut. The DGGE analysis method is used for analyzing bacterial diversity in sauerkraut.
Description
Technical field
The present invention relates to the analytical procedure of Phylogenetic diversity of bacteria in a kind of spontaneous fermentation sauerkraut
Background technology
Sauerkraut is the fermented vegetables products that fresh Chinese cabbage salt solution is formed after brewed for some time in encloses container, the sauerkraut of spontaneous fermentation has been given its unique local flavor, the fermentation mode of this uniqueness has determined wherein microbiotic diversity, in sauerkraut fermenting process, can produce various bacteria, the fermentative action of the different times control sauerkraut that these bacteriums are fermented at sauerkraut respectively, makes sauerkraut produce unique local flavor.
The research of people to bacterial population complicated in sauerkraut, the traditional Fen Li ﹑ Purification and Characterization method of many employings, not only numerous and diverse consuming time, and it is larger affected by culture condition and subjective factor, can not reflect the multifarious truth of bacterium in sauerkraut, result accuracy and poor reliability, therefore, traditional cultural method can not comprehensively and objectively reflect the information such as population structure, Phylogenetic diversity of bacteria of bacterium in sauerkraut.
What DGGE (denatured gradient gel electrophoresis) denaturing gradient gel electrophoresis was the people such as Fisher and Lerman in the invention of phase early 1980s at first is mainly used to detect the point mutation in DNA fragmentation, this technology was widely used in the every field of Molecular Ecology of Microbiology research afterwards, had developed into one of main molecules biological method of microorganisms structure of community at present.Principle be double chain DNA molecule in the time of general polyacrylamide gel electrophoresis, its migratory behaviour is decided by its molecular size and electric charge.The DNA fragmentation of different lengths can be distinguished, but the migratory behaviour of the DNA fragmentation of same length in glue is the same, therefore can not be distinguished.DGGE technology is on general polyacrylamide gel basis, denaturing agent (urea and methane amide) gradient or thermograde are added, not homotactic DNA fragmentation is unwinding under corresponding denaturing agent concentration separately, there is the variation of space structure, cause electrophoretic velocity sharply to decline, thus can be same length but the different DNA fragmentation of sequence make a distinction.
Summary of the invention
The DGGE analytical procedure of Phylogenetic diversity of bacteria in spontaneous fermentation sauerkraut in northeast of the present invention, carry out according to the following steps:
1, the pre-treatment of sauerkraut sample and microorganism collection.
2, adopt FastPrep to carry out the rapid extraction of total DNA of microorganism in sauerkraut sample in conjunction with CTAB method.
3, taking total DNA of extracting microorganism as template, adopt 16S rRNA gene V3 district to there is specific primer pair, carry out the pcr amplification of bacterial 16 S rRNA, by the product 8%(mass/volume of PCR reaction) agarose gel electrophoresis detection.
4, PCR product is carried out to DGGE electrophoretic analysis according to following condition: selecting denaturing agent concentration gradient is 35%～55%, now electrophoretic buffer is preheated to 60 DEG C, with 150V voltage prerunning 10min, then adds the PCR product of 15 μ l to each well, under 150V voltage, electrophoresis 7h.
5, electrophoresis finish rear to DGGE product dye, scanner imaging, obtain DGGE collection of illustrative plates.Through glue map analysis, mark specific band on sauerkraut sample bacterium DGGE collection of illustrative plates, cuts glue and reclaims.
6, the DNA band reclaiming need re-start pcr amplification, then product is checked order, and the sequence recording is carried out to Blast comparison at ncbi database, determines the ownership of bacterium, completes the DGGE analytical procedure of Phylogenetic diversity of bacteria in northeast spontaneous fermentation sauerkraut.
Positively effect of the present invention:
Population structure, the Phylogenetic diversity of bacteria of bacterium in the comprehensive and accurate reflection sauerkraut of present method energy, compared with pure culture method, the advantages such as it is easy and simple to handle, quick, reproducible that the method has, simultaneously can also a large amount of samples of comparative analysis fast, therefore the bacterial flora difference that it can the comparative analysis same sauerkraut different fermentations stage, also can study the bacterial flora difference of different spontaneous fermentation sauerkrauts.Adopt DGGE to analyze and more than 1% bacterium of relative content in flora can be detected, and can analyze microorganism collection of illustrative plates.
Brief description of the drawings
Fig. 1 is the agarose gel electrophoresis figure of sauerkraut fermentation broth sample pcr amplification.
Fig. 2 is the PCR-DGGE collection of illustrative plates of sauerkraut fermentation broth sample bacteria total DNA.
Embodiment
In order to understand better the present invention, specifically tell about specific embodiment of the invention step below:
1. the pre-treatment of sauerkraut fermentation broth sample:
Sample is the natural sauerkraut sample that the Northeast gathers arbitrarily, sample multiple spot is taken from sauerkraut fermentation juice, after mixing, in 5mL sauerkraut fermentation broth sample, add 5mL PBS damping fluid, vortex concussion 30s, the then centrifugal 5min of 350rpm, collect supernatant, after the centrifugal 5min of 12000rpm, abandon supernatant, collect thalline, in precipitation, add 800 μ L TE damping fluid back dissolvings, for the extraction of microorganism total DNA.
2. the extraction of microorganism total DNA in sauerkraut fermented liquid
Adopt Fast Prep(quick instrument for extracting nucleic acid) carry out the rapid extraction of microorganism total DNA in conjunction with CTAB method.Concrete steps are as follows:
(1) get sample described in pretreated step 1 in the broken pipe of 2mL, add 0.5g granulated glass sphere, be placed in FastPrep nucleic acid Rapid extraction instrument, the 5.5m ﹒ s-1 45s that vibrate, quick lysate sample.
(2) cracking: add 10%(mass/volume) SDS lysate 60 μ l, mix the centrifugal 10min of rear 12000rpm, be transferred in the aseptic centrifuge tube of 1.5ml, add the Nacl of 100 μ l5mol/l, the CTAB of 80ul10mol/l, 65 DEG C of water-bath 20min.
(3) extracting: the phenol that adds 1ml: chloroform: primary isoamyl alcohol=25:24:1(volume ratio), softly mix, leave standstill 1min, the centrifugal 10min of 12000rpm, get supernatant liquor and again carry out extracting, add the chloroform of 1ml: primary isoamyl alcohol=24:1(volume ratio), the centrifugal 10min of 12000rpm, gets supernatant liquor to aseptic 1.5ml centrifuge tube.
(4) precipitation: add 500 μ l ice Virahols, the NaAC of 50 μ l3mol/L, puts upside down, leaves standstill, and mixes, and places 30min for-20 DEG C, precipitation thalline, the centrifugal 10min of 12000rpm.
(5) rinsing and air-dry: abandon supernatant liquor, add 500 μ l70%(volume ratios) absolute ethanol washing precipitation, mix gently, the centrifugal 10min of 12000rpm, abandons supernatant, is placed in aseptic operating platform vertically-supplying air and dries up, and adds 30 μ lTE dissolution precipitations, and-20 DEG C save backup.
3.PCR amplification
Get that 1.5 μ l extract total DNA be template, adopt 16S rRNA gene V3 district to there is specific primer pair and carry out the pcr amplification of 16S rRNA.Primer sequence is V3F+GC:(5 '-CGCCC GCCGCGCGCG GCGGG CGGGG CGGGGGC CCTACGG GAGGC AGCAG-3 ') and V3R:(5 '-ATTACC GCG GCT GCT GG-3 ').
Amplification reaction system is 50 μ l, is made up of following ingredients:
Reaction parameter: 95 DEG C of denaturation 4min, 30 circulations comprise 95 DEG C of sex change 1min, 55 DEG C of annealing 45s, 72 DEG C are extended 1min, and last 72 DEG C are extended 7min, 4 DEG C of insulations.The product of PCR reaction detects with 8% agarose gel electrophoresis.
4.DGGE analyzes
DGGE gum concentration is 8%(mass/volume) polyacrylamide gel, denaturing agent concentration is 35%～55%, adopt the DCode electrophoresis apparatus of Bio-Rad company to carry out electrophoresis, electrophoretic buffer is preheated to 60 DEG C, with 150V voltage prerunning 10min, then add the PCR product of 15 μ l to each well, under 150V voltage, electrophoresis 7h.(see figure 1)
5, after electrophoresis, utilize argentation to dye to gel, concrete staining procedure is as follows:
(1) DGGE glue stationary liquid: 450ml H
2o, 50ml dehydrated alcohol, 4ml Glacial acetic acid, fixing 15min;
(2) silver-colored dye liquor: 400ml H
2o, 4ml Silver Nitrate, 200 μ l formaldehyde, dyeing 10min, then uses a small amount of deionized water rinsing;
(3) nitrite ion: 600ml H
2o, sodium hydroxide 9g, 600 μ l formaldehyde, treat that can take out appears in band;
(4) last stop buffer: 496ml H
2o, 4ml Glacial acetic acid, termination reaction.
The gel that silver has been dyed scanner imaging, obtains DGGE collection of illustrative plates (see figure 2), and the collection of illustrative plates after above-mentioned scanning is analyzed, and mark specific band on the DGGE collection of illustrative plates of sauerkraut sample DNA of bacteria, to its numbering, cutting, recovery.
6, the DNA band reclaiming need re-start pcr amplification, then product is checked order, check order in conjunction with 454 pairs of end sequencings (Paired-End) and Illumina end pairing order-checking (Mate-Pair Sequencing) technology, the sequence recording is carried out to Blast comparison at ncbi database, determine the ownership of bacterium, and login ncbi database and carry out the comparative analysis of Blast homology, comparison result is in table 1.
Table 1.DGGE collection of illustrative plates separation of bacterial V3 region sequence similarity comparative result
From result, plant lactobacillus Lactobacillus plantarum, short lactobacillus Lactobacillusbrevis, lactobacillus sake Lactobacillus sakei and lactobacillus curvatus Lactobacillus curvatus, four kinds of bacterium are the dominant microflora in northeast spontaneous fermentation sauerkraut fermented liquid.
Agricultural University Of Shenyang
The DGGE analytical procedure of Phylogenetic diversity of bacteria in northeast spontaneous fermentation sauerkraut
3
1
17
DNA artificial sequence
CCT?ACG?GGA?GGC?AGC?AG17
PCR primer V3F sequence.
2
17
DNA artificial sequence
ATT?ACC?GCG?GCT?GCT?GG17
PCR primer V3R sequence.
3
32
DNA artificial sequence
CGC?CCG?CCG?CGC?GCG?GCG?GGC?GGG?GCG?GGG?GC32
5 ' section of base sequence adding of primer V3F
Claims (6)
1. a DGGE analytical procedure for Phylogenetic diversity of bacteria in northeast spontaneous fermentation sauerkraut, is characterized in that the method carries out according to the following steps:
(1), select sauerkraut sample, sample is carried out to pre-treatment and microorganism collection;
(2), adopt FastPrep to carry out the rapid extraction of microorganism total DNA in sauerkraut sample in conjunction with CTAB method;
(3), taking total DNA of the microorganism of extracting as template, adopt 16S rRNA gene V3 district to there is specific primer pair. carry out the pcr amplification of bacterial 16 S rRNA, the product of PCR reaction detected with agarose gel electrophoresis;
(4), PCR product is carried out to DGGE electrophoretic analysis;
(5), electrophoresis finish rear to DGGE product dye, scanner imaging, obtain DGGE collection of illustrative plates,
Mark specific band on sauerkraut sample bacterium DGGE collection of illustrative plates, cuts glue and reclaims;
(6) the DNA band, reclaiming need re-start pcr amplification, then product is checked order, the sequence results recording is carried out to the comparative analysis of Blast homology at ncbi database, determine the ownership of bacterium, complete the DGGE analytical procedure of Phylogenetic diversity of bacteria in northeast spontaneous fermentation sauerkraut.
2. method according to claim 1, is characterized in that in step (1), and sample is the natural sauerkraut sample that the Northeast gathers arbitrarily, and sample multiple spot is taken from sauerkraut fermentation juice, carries out pre-treatment after mixing.
3. method according to claim 1, is characterized in that in step (1), and the sauerkraut sample gathering is carried out to pre-treatment, collect thalline, concrete steps are as follows: in 5mL sauerkraut fermentation broth sample, add 5mL PBS damping fluid, vortex concussion 30s, the then centrifugal 5min of 350rpm, collect supernatant, after the centrifugal 5min of 12000rpm, abandon supernatant, collect thalline, in precipitation, add 800 μ L TE damping fluid back dissolvings, for the extraction of microorganism total DNA.
4. method according to claim 1, is characterized in that in step (2), adopts the quick instrument for extracting nucleic acid of Fast Prep to carry out the rapid extraction of microorganism total DNA in sauerkraut sample in conjunction with CTAB method, and concrete steps are as follows:
1. get sample described in pretreated step (1) in the broken pipe of 2mL, add 0.5g granulated glass sphere, be placed in Fast Prep nucleic acid Rapid extraction instrument, the 5.5m ﹒ s-1 45s that vibrate, quick lysate sample;
2. cracking: add 10%(mass/volume) SDS lysate 60 μ l, mix the centrifugal 10min of rear 12000rpm, be transferred in the aseptic centrifuge tube of 1.5ml, add the Nacl of 100 μ l 5mol/l, the CTAB of 80ul10mol/l, 65 DEG C of water-bath 20min;
3. extracting: the phenol that adds 1ml: chloroform: primary isoamyl alcohol=25:24:1, softly mix, leave standstill 1min; the centrifugal 10min of 12000rpm, gets supernatant liquor and again carries out extracting, adds the chloroform of 1ml: primary isoamyl alcohol=24:1; the centrifugal 10min of 12000rpm, gets supernatant liquor to aseptic 1.5ml centrifuge tube;
4. precipitation: add 500 μ l ice Virahols, the NaAC of 50 μ l 3mol/L, puts upside down, leaves standstill, and mixes, and places 30min for-20 DEG C, precipitation thalline, the centrifugal 10min of 12000rpm;
5. rinsing and air-dry: abandon supernatant liquor, add 500 μ l70%(actively than) absolute ethanol washing precipitation, the centrifugal 10min of 12000rpm, abandons supernatant, is placed in aseptic operating platform vertically-supplying air and dries up, and adds 30 μ lTE dissolution precipitations ,-20 DEG C save backup.
5. method according to claim 1, is characterized in that the reaction system of pcr amplification in step 3 is wherein the reaction system of 50 μ l, is made up of following ingredients:
Pcr amplification condition is 95 DEG C of denaturation 4min, and 30 circulations comprise 95 DEG C of sex change 1min, 55 DEG C of annealing 45s, and 72 DEG C are extended 1min, and last 72 DEG C are extended 7min, 4 DEG C of insulations.
6. method according to claim 1, is characterized in that the detection in Gene Mutation system that in step 4 wherein, electrophoresis apparatus is Dcode, and deposition condition is voltage 150V, electrophoresis 7h at 60 DEG C, and denatured gradient is 35%～55%.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106811538A (en) * | 2017-03-21 | 2017-06-09 | 舟山市食品药品检验检测研究院 | The PCR DGGE analysis methods of specific spoilage organisms during a kind of peeled shrimp cryopreservation |
| CN111621448A (en) * | 2020-07-03 | 2020-09-04 | 沈阳农业大学 | Bacillus belgii SN-1 and method for producing exopolysaccharides through fermentation of bacillus belgii SN-1 |
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| CN101654705A (en) * | 2009-09-09 | 2010-02-24 | 南昌大学 | Application of DGGE marker of lactobacillus and bifidobacteria to flora monitoring |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106811538A (en) * | 2017-03-21 | 2017-06-09 | 舟山市食品药品检验检测研究院 | The PCR DGGE analysis methods of specific spoilage organisms during a kind of peeled shrimp cryopreservation |
| CN111621448A (en) * | 2020-07-03 | 2020-09-04 | 沈阳农业大学 | Bacillus belgii SN-1 and method for producing exopolysaccharides through fermentation of bacillus belgii SN-1 |
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Application publication date: 20140730 |