CN105385762A - Method for rapidly identifying bifidobacterium - Google Patents

Method for rapidly identifying bifidobacterium Download PDF

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CN105385762A
CN105385762A CN201510917117.0A CN201510917117A CN105385762A CN 105385762 A CN105385762 A CN 105385762A CN 201510917117 A CN201510917117 A CN 201510917117A CN 105385762 A CN105385762 A CN 105385762A
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glue
bifidus bacillus
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pcr
electrophoresis
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陈卫
赵国忠
韩俊燕
印伯星
房东升
张秋香
张白曦
田丰伟
范大明
刘小鸣
王刚
郭敏
赵建新
张灏
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YANGZHOU YANGDA KUANGYUANG DAIRY INDUSTRY Co Ltd
Jiangnan University
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YANGZHOU YANGDA KUANGYUANG DAIRY INDUSTRY Co Ltd
Jiangnan University
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    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

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Abstract

The invention relates to a method for rapidly identifying a bifidobacterium. The strips of an unknown strain and a standard strain are compared through adopting a PCR-DGGE technology to identify the unknown bifidobacterium to a species or subspecies level. The method can be used to rapidly and accurately identifying the bifidobacterium to the species or subspecies level, and provides a reliable way for separation, identification and further researches of bifidobacteria; and one variable region of a bifidobacterium 16S rDNA can be amplified through the method, strip comparison between the fingerprint of the obtained bifidobacterium and the standard fingerprint of the known bifidobacterium is carried out to rapidly identify the bifidobacterium to the species or subspecies level.

Description

A kind of method of Rapid Identification of Bifidobacteria
Technical field
The invention belongs to microbial technology field, relate to bifidus bacillus, especially a kind of molecular biology method of Rapid Identification of Bifidobacteria.
Background technology
Bifidus bacillus is the gram positive bacterium of a class anaerobism, and according to the classification of the outstanding Bacteria Identification handbook of uncle, bifidus bacillus belongs to actinomycetales Actinomy cetaceae genus bifidobacterium.Bifidus bacillus is the rhabdos that appearance change is very large, and the general feature of bacterial strain is Y type in bifurcated or V-type, and club-like or spoon-shaped type.1899, first French Pasteur's Institute personnel isolated the first strain bifidus bacillus from breast-fed infant's ight soil, and increasing scientist and investigators start the research work to bifidus bacillus afterwards.And practice shows, bifidus bacillus is present in the class probiotics in people and other animal intestinal, and the quantity of bifidus bacillus in human intestinal, the abundantest at infant's content in period, with advancing age, ratio reduces gradually.Surely grow the bifidus bacillus in human intestinal, suppressing pernicious bacteria, maintain intestinal microflora balance, promote intestines peristalsis, improve constipation, alleviate caused by microbiotic diarrhoea, improve in host immunity etc. and play an important role.
Along with going deep into of studying bifidus bacillus, and the exploitation to bifidus bacillus series products, how obtaining the bifidobacterium strains with potential probiotic properties fast becomes problem demanding prompt solution.And for the qualification of bifidus bacillus, be wherein indispensable integral part.At present, for the strain identification of bifidus bacillus, it is the conventional identification method of carrying out with physio-biochemical characteristics (mainly carbohydrate metabolism is different) based on morphology that conventional method mainly contains two kinds: one; Two is based on molecular biology method, measures the gene of bacterial strain, and wherein the most frequently used is the 16SrDNA fragment of pcr amplification bifidus bacillus, the level to be identified by bifidus bacillus kind.But all there is certain inevitable defective to a certain extent in these two kinds of methods.Traditional morphology and Physiology and biochemistry authentication method, due to the form polytropy of bifidus bacillus and the nuance of metabolism substrate, be difficult to bifidus bacillus be identified the level of planting.And it is large to there is workload, the shortcoming of bothersome effort.And adopt 16SrDNA fragment to carry out the method checked order, although can precise Identification to kind, there is the shortcoming that program is loaded down with trivial details.
The principle of work of denaturing gradient gel electrophoresis (DGGE) is, according to the melting properties of DNA, the concentration of denaturing agent needed for the duplex DNA generation sex change of different based composition is different.The double-stranded DNA of mixing is when carrying out the electrophoresis that denaturant concentration linearly changes, the DNA of identical based composition unwinds in the position of same denaturant concentration, the DNA of different based composition unwinds in the position of different denaturation agent concentration, rest on the different positions in gel due to the difference of rate of migration, thus the DNA fragmentation of mixing is separated.This technology can be separated only having the DNA fragmentation of a pair base difference.And the V3 district of the 16SrDNA of bifidus bacillus is hypervariable region, bifidus bacillus not of the same race can be identified out by DGGE means in theory.
By retrieval, find patent publication us as relevant to patent application of the present invention in next chapter:
A kind of method (CN102108400A) identifying bifidus bacillus, relates to a kind of method identifying bifidus bacillus, is specifically related to the molecular biology method of a kind of qualification bifidus bacillus newly.The method by the pcr amplification of the upper two sections of target gene fragments of bifidus bacillus 16SrDNA and pyruvate kinase encoding gene the preceding paragraph target gene fragment and sequencing analysis, the level that bifidobacterium strains can be identified kind.The present invention by provide for bifidus bacillus qualification a kind of more accurately, be easier to the practical approach that operates, be conducive to classification and the research work of bifidobacterium strains.
By contrast, there are the different of essence in patent application of the present invention and above-mentioned patent publication us.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art part, a kind of method identifying bifidus bacillus is quickly and accurately provided, bifidus bacillus can to identify kind or the level of subspecies by the method, for bifidus bacillus isolation identification and further research a kind of reliable means is provided.
The technical solution adopted for the present invention to solve the technical problems is:
A method for Rapid Identification of Bifidobacteria, adopts the method for PCR-DGGE, is compared by the band of unknown strains and reference culture, unknown bifidus bacillus is identified the level of kind or subspecies.
And step is as follows:
(1) extract the genome of the bifidus bacillus not of the same race of known sequence;
(2) adopt appropriate primer pair, carry out the V3 district of the 16SrDNA of pcr amplification list bacterium;
(3) adopt denaturing gradient gel electrophoresis method to build standard finger-print;
(4) the bifidus bacillus band of the unknown and standard band are compared, determine the kind of unknown bifidus bacillus.
And, described step (1) in the bifidus bacillus of known sequence be bifidobacterium breve, bifidumbacterium bifidum, animal bifidobacteria, bifidobacterium adolescentis, the long subspecies of bifidus longum bb and bifidus longum bb baby subspecies.
And the described step (1) genomic method of middle extraction bifidus bacillus is phenol-chloroform extraction process.
And described step (2) middle primer pair is 338f-GC and 518r.
And the base sequence of described primer 338f-GC is 5 '-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGACTCCTACGGGAGGCA GCAG-3 '; The base sequence of primer 518r is 5 '-ATTACCGCGGCTGCTGG-3 '.
And, described step (2) in the amplification condition of PCR be: 95 DEG C, 5min; 94 DEG C, 45s, 65 DEG C, 45s, each cycle down 0.5 DEG C, 72 DEG C, 30s, 20 circulations; 94 DEG C, 50s, 55 DEG C of 55s, 72 DEG C of 30s, 15 circulations; 72 DEG C of 10min.
And the described step glue that (3) middle denaturing gradient gel electrophoresis method is used is the polyacrylamide of 30% ~ 70%.
And, described step (3) in the deposition condition of denaturing gradient gel electrophoresis method be: 120V, 30min; 80V, 12h; Damping fluid is 1 × TAE; Temperature is 60 DEG C.
And concrete steps are as follows:
(1) substratum and culture condition
Substratum: MRS+5 ‰ halfcystine, 121 DEG C of sterilizing 15min; Culture condition: 37 DEG C, cultivates 24h in anaerobism workstation, obtains bacteria suspension;
(2) genome extracts
(1) get the bacteria suspension 1mL of overnight incubation in 1.5mL centrifuge tube, the centrifugal 2min of 10000rpm, abandons supernatant and obtains thalline;
(2), after washing thalline with the piping and druming of 1mL sterilized water, the centrifugal 2min of 10000rpm, abandons supernatant and obtains thalline;
(3) in thalline, add 200uLSDS lysate, 80 DEG C of water-bath 30min, obtain cellular lysate liquid;
(4) add phenol-chloroform solution 200uL in cellular lysate liquid, wherein the moiety of phenol-chloroform solution and volume ratio are: the saturated phenol of Tris: chloroform: primary isoamyl alcohol=25:24:1, and after putting upside down mixing, the centrifugal 5-10min of 12000rpm, gets supernatant 200uL;
(5) add 400uL ice ethanol or ice Virahol in 200uL supernatant ,-20 DEG C of centrifugal 5-10min of standing 1h, 12000rpm, abandon supernatant;
(6) add the resuspended precipitation of ice ethanol that 500uL percent by volume is 70%, the centrifugal 1-3min of 12000rpm, abandons supernatant;
(7) 60 DEG C of oven for drying, or naturally dry, obtain precipitation;
(8) 50uLddH 2the heavy molten precipitation of O, in order to PCR;
(3) 16SrDNAV3 district primer
Upstream primer 338f-GC, base sequence: SEQ1 (5 '-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGACTCCTACGGGAGGCA GCAG-3 ');
Downstream primer 518r, base sequence: SEQ2 (5 '-ATTACCGCGGCTGCTGG-3 ');
(4) PCR reaction system 50 μ L:
10 × Taqbuffer, 5 μ L; DNTP, 5 μ L; 338f-GC20 μM, 0.5 μ L; 518r20 μM, 0.5 μ L; Taq enzyme, 0.5 μ L; DNA profiling, 0.5 μ L; ddH 2o, 38 μ L;
(5) PCR response procedures
95 DEG C, 5min; 94 DEG C, 45s, 65 DEG C, each cycle down of 45s 0.5 DEG C, 72 DEG C, 30s, 20 circulations; 94 DEG C, 50s, 55 DEG C of 55s, 72 DEG C 30s15 circulation; 72 DEG C, keep 10min;
(6) agarose electrophoresis
(1) prepare the sepharose of mass percent 1%: every large glue 40mL, take 0.4g agarose and be placed in Erlenmeyer flask, add the 0.5 × TBE of 40mL, microwave-oven-heating boils to agarose and all melts, shake up, treat that solution is chilled to non-scald on hand, add 4 μ L10000 × nucleic acid dye, pour into after mixing in mould, solidify more than 30min;
(2), after genome DNA sample/PCR primer sample being mixed with 10 × Loadingbuffer, add in sample cell;
(3) switch on power, 120V, runs 30min;
(4), after electrophoresis terminates, take pictures with gel imaging instrument;
(7) operation of denaturing gradient gel electrophoresis
(1) solution preparation
1. 50 × TAE electrophoretic buffer: tris242g, Na 2eDTA2H 2o37.2g, adds 800mL deionized water, fully mixes, and adds 57.1mL Glacial acetic acid, fully mixes, and adds deionized water to 1L, mixing;
2. 0% denaturing agent 100mL: the acrylamide of mass percent 40%, 20mL; 50 × TAE electrophoretic buffer, 2mL; DH 2o, 78mL, cross 0.45 μm of filter membrane, ultrasonic degas 10-15min, is placed in brown reagent bottle;
3. 100% denaturing agent 100mL: the acrylamide of mass percent 40%, 20mL; 50 × TAE electrophoretic buffer, 2mL; Methane amide, 40mL; Urea, 42g; DH 2o, to 100mL, cross 0.45 μm of filter membrane, ultrasonic degas 10-15min, is placed in brown reagent bottle, must be heavy molten in warm water bath before using;
4. 10% ammonium persulphate: 0.1g ammonium persulphate is dissolved in 1mLdH 2in O ,-20 DEG C of storages;
(2) glue
1. the sheet glass caused not of uniform size for two panels is cleaned up, dry;
2. two spacer bars are placed in structural glass plate both sides of the edge, outward flange is mutually neat with glass plate edge, the groove of spacer bar upward, pieces of glass plate is placed on spacer bar, and align with it, and this sandwich structure clip is fixed, hold up the rear middle calibration plate that inserts and correct, both sides retaining screw screws;
3. sponge pad is fixed on gum-making rack, sandwich structure is fixed on gum-making rack together with clip, adds deionized water, test leakage, determine after not leaking, water outwelled and with filter paper, residual water blotted;
4. the gel of 30% and 70% is prepared respectively, wherein, the glue of 30%: 0% denaturing agent, 14mL; 100% denaturing agent, 6mL; Tetramethyl Ethylene Diamine, 10 μ L; 10% ammonium persulphate, 80 μ L;
The glue of 70%: 0% denaturing agent, 6mL; 100% denaturing agent, 14mL; Tetramethyl Ethylene Diamine, 10 μ L; 10% ammonium persulphate, 80 μ L;
5. after two syringes suck the gel of 30% and 70% respectively, discharge bubble, enter the lower concentration of glue and high density is fixed on gradient transfer system according to top, Y tube in connection, carefully drives bubble away;
6. glass core position aimed at by syringe needle, and pin hole is towards structural glass plate, and softly also stably rotating cam transmits solution, remains a constant speed, makes solution constant speed be poured in the gel slab of sandwich style;
7. carefully insert comb, allow gel polymerisation one hour, the equipment that rapid cleanup is finished, especially wash liquid residual in Y tube off, in order to avoid blocking;
8. add 7L1 × TAE buffered soln in electrophoresis chamber, it is diluted by 50 × TAE buffered soln and obtains, and open electrophoresis control device, preheating electrophoretic buffer is to 60 DEG C;
9. pull out away comb after polymerization, put into by glue in electrophoresis chamber, cleaning loading wells, covers temperature-control device and makes temperature rise to 60 DEG C;
(3) loading: after having added sample loading buffer in PCR primer, each glue hole adds 20 μ L;
(4) electrophoresis: 80V, 12h after 120V, 30min, the principle followed is voltage V × time h is 1000;
(5) dye
1. after electrophoresis, first push one piece of sheet glass aside, then together with another block sheet glass, glue is put into pallet, glue upward;
2. GelRed diluent: 30mL water+3 μ L10000 × GelRed mixes, and to obtain final product, to keep in Dark Place;
3. being divided by GelRed diluent with the pipettor of 1mL is added on glue 3 times, and each 10mL, must cover loading wells corresponding zone, add rear 10min of Denging at every turn, pallet palpus cover lid lucifuge;
4. add deionized water and do not have glue, lucifuge 30min;
5. rock pallet gently, make water enter between glue and sheet glass, slowly make it be separated, take out sheet glass;
(6) develop: ultraviolet development under gel imaging instrument, take pictures;
(8) the DGGE qualification of unknown bifidus bacillus
According to the operation steps of embodiment (two) to (six), obtain the PCR primer in the 16SrDNAV3 district of unknown bifidus bacillus;
Using the hybrid standard finger printing of bifidus bacillus as Marker, the bifidus bacillus of the unknown is identified.
The advantage that the present invention obtains and beneficial effect are:
1, bifidus bacillus can be identified the level of kind or subspecies by present method quickly and accurately, for bifidus bacillus isolation identification and further research a kind of reliable means is provided, the method only amplifies a certain variable region of bifidus bacillus 16SrDNA, carry out band comparison with the standard finger-print of known bifidus bacillus, rapidly bifidus bacillus can be identified the level of kind or subspecies.
2, the invention provides a kind of new approaches of Rapid Identification of Bifidobacteria, namely a certain hypervariable region (V3, V4 or V6-V8) of the method for PCR-DGGE amplification bifidus bacillus 16SrDNA is utilized, utilize the difference of this region base sequence to be separated by the bifidus bacillus of different Known Species and make Marker, the bifidus bacillus that qualification is unknown, theoretically, the method can identify all different types of bifidus bacilluss.
3, present invention provides the new approaches of a kind of bacterium of other kinds of Rapid identification, such as Bacterium lacticum etc., theoretically, as long as not there is the difference of base pair in a certain variable region of the 16SrDNA of Bacterium lacticum of the same race etc., just can amplify this region according to the thinking of this invention, the Marker producing Bacterium lacticum equally identifies Bacterium lacticum not of the same race.
Accompanying drawing explanation
Fig. 1 is the agarose gel electrophoresis figure of 6 kinds of bifidus bacillus genomic dnas in the embodiment of the present invention;
Fig. 2 is the agarose gel electrophoresis figure in 6 kinds of bifidus bacillus 16SrDNAV3 districts in the embodiment of the present invention;
Fig. 3 is the DGGE standard finger-print in 6 kinds of bifidus bacillus 16SrDNAV3 districts in the embodiment of the present invention;
Fig. 4 is the DGGE qualification result figure in unknown bifidus bacillus 16SrDNAV3 district in the embodiment of the present invention.
Embodiment
Below in conjunction with embodiment, the present invention is further described; Following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
The raw material used in the present invention, if no special instructions, is conventional commercially available prod; The method used in the present invention, if no special instructions, is the ordinary method of this area.
The invention provides a kind of method of Rapid Identification of Bifidobacteria, main technological route is exactly: the first V3 district of the 16SrDNA of the bifidus bacillus that pcr amplification gene order is known; Then the standard finger-print of bifidus bacillus is built by DGGE; Finally the band in the V3 district of the bifidus bacillus 16SrDNA of the unknown and standard band are compared, determine the kind of unknown bifidus bacillus.
Embodiment 1
A method for Rapid Identification of Bifidobacteria, is characterized in that: the method adopting PCR-DGGE, is compared by the band of unknown strains and reference culture, unknown bifidus bacillus is identified the level of kind or subspecies;
Step is as follows:
(1) extract the genome of the bifidus bacillus not of the same race of known sequence;
(2) adopt appropriate primer pair, carry out the V3 district of the 16SrDNA of pcr amplification list bacterium;
(3) adopt denaturing gradient gel electrophoresis method to build standard finger-print;
(4) the bifidus bacillus band of the unknown and standard band are compared, determine the kind of unknown bifidus bacillus.
Preferentially, described step (1) in the bifidus bacillus of known sequence be bifidobacterium breve, bifidumbacterium bifidum, animal bifidobacteria, bifidobacterium adolescentis, the long subspecies of bifidus longum bb and bifidus longum bb baby subspecies;
The described step (1) genomic method of middle extraction bifidus bacillus is phenol-chloroform extraction process;
Described step (2) middle primer pair is 338f-GC and 518r;
The base sequence of described primer 338f-GC is SEQ1; The base sequence of primer 518r is SEQ2;
Described step (2) in the amplification condition of PCR be: 95 DEG C, 5min; 94 DEG C, 45s, 65 DEG C, 45s, each cycle down 0.5 DEG C, 72 DEG C, 30s, 20 circulations; 94 DEG C, 50s, 55 DEG C of 55s, 72 DEG C of 30s, 15 circulations; 72 DEG C of 10min;
The described step glue that (3) middle denaturing gradient gel electrophoresis method is used is the polyacrylamide of 30% ~ 70%;
Described step (3) in the deposition condition of denaturing gradient gel electrophoresis method be: 120V, 30min; 80V, 12h; Damping fluid is 1 × TAE; Temperature is 60 DEG C.
Embodiment 2
A method for Rapid Identification of Bifidobacteria, step is as follows:
(1) substratum and culture condition
Substratum: MRS+5 ‰ halfcystine, 121 DEG C of sterilizing 15min; Culture condition: 37 DEG C, the bifidus bacillus not of the same race of getting known sequence cultivates 24h in anaerobism workstation.
(2) genome extracts
1. get the bacteria suspension 1mL of overnight incubation in 1.5mL centrifuge tube, the centrifugal 2min of 10000rpm, abandons supernatant and obtains thalline;
2., after washing thalline with the piping and druming of 1mL sterilized water, the centrifugal 2min of 10000rpm, abandons supernatant and obtains thalline;
3. add 200uLSDS lysate, 80 DEG C of water-bath 30min;
4. add phenol-chloroform solution 200uL in cellular lysate liquid, wherein the moiety of phenol-chloroform solution and volume ratio are: the saturated phenol of Tris: chloroform: primary isoamyl alcohol=25:24:1, and after putting upside down mixing, the centrifugal 5-10min of 12000rpm, gets supernatant 200uL;
5. add 400uL ice ethanol or ice Virahol in 200uL supernatant ,-20 DEG C of centrifugal 5-10min of standing 1h, 12000rpm, abandon supernatant;
6. add the resuspended precipitation of 500uL70% (percent by volume) ice ethanol, the centrifugal 1-3min of 12000rpm, abandons supernatant;
7.60 DEG C of oven for drying, or naturally dry;
8.50uLddH 2the heavy molten precipitation of O is in order to PCR.
(3) 16SrDNAV3 district primer
Upstream primer 338f-GC, base sequence: SEQ1:5 '-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGACTCCTACGGGAGGCA GCAG-3 ';
Downstream primer 518r, base sequence: SEQ2:5 '-ATTACCGCGGCTGCTGG-3 '.
(4) PCR reaction system (50 μ L)
10 × Taqbuffer, 5 μ L; DNTP, 5 μ L; 338f-GC (20 μMs), 0.5 μ L; 518r (20 μMs), 0.5 μ L; Taq enzyme, 0.5 μ L; DNA profiling, 0.5 μ L; ddH 2o, 38 μ L.
(5) PCR response procedures
95 DEG C, 5min; 94 DEG C, 45s, 65 DEG C, 45s (each cycle down 0.5 DEG C), 72 DEG C, 30s, 20 circulations; 94 DEG C, 50s, 55 DEG C of 55s, 72 DEG C of 30s (15 circulations); 72 DEG C, keep 10min.
(6) agarose electrophoresis
1. prepare the sepharose (large glue 40mL) of 1%: take 0.4g agarose and be placed in Erlenmeyer flask, add the 0.5 × TBE of 40mL.Microwave-oven-heating boils and all melts to agarose, shakes up, and treats that solution is chilled to non-scald on hand, add 4 μ L10000 × nucleic acid dye, pour into after mixing in mould, solidify more than 30min;
2., after genome DNA sample/PCR primer sample being mixed with 10 × Loadingbuffer, add in sample cell;
3. switch on power, 120V, runs 30min;
4., after electrophoresis terminates, take pictures with gel imaging instrument.
Wherein, as shown in Figure 1, known genomic dna has been extracted 6 kinds of genomic agarose gel electrophoresis figure of bifidus bacillus well; The agarose gel electrophoresis figure in 6 kinds of bifidus bacillus 16SrDNAV3 districts as shown in Figure 2, known single stripe size at about 230bp, for the purpose of the length of fragment.
(7) DGGE operation steps
1. solution preparation
(1) 50 × TAE electrophoretic buffer: tris242g, Na 2eDTA2H 2o37.2g, adds 800mL deionized water, fully mixes, and adds 57.1mL Glacial acetic acid, fully mixes, and adds deionized water to 1L, mixing.
(2) 0% denaturing agents (100mL): 40%Acrylamide/Bis acrylamide, 20mL; 50 × TAE electrophoretic buffer, 2mL; DH 2o, 78mL.Cross 0.45 μm of filter membrane, ultrasonic degas 10-15min, be placed in brown reagent bottle.
(3) 100% denaturing agents (100mL): 40%Acrylamide/Bis acrylamide, 20mL; 50 × TAE electrophoretic buffer, 2mL; Formamide (deionized) methane amide, 40mL; Urea urea, 42g; DH 2o, to100mL.Cross 0.45 μm of filter membrane, ultrasonic degas 10-15min, be placed in brown reagent bottle.Must be heavy molten in warm water bath before using.
(4) 10%AmmoniumPersulfate ammonium persulphate: 0.1g ammonium persulphate is dissolved in 1mLdH 2in O ,-20 DEG C of storages.
2. glue
(1) two glass sheets are cleaned up, dry.
(2) two spacer bars are placed in structural glass plate both sides of the edge, outward flange is mutually neat with glass plate edge, the groove of spacer bar upward, pieces of glass plate is placed on spacer bar, and align with it, and this " sandwich " is fixed with clip, hold up the rear middle calibration plate that inserts and correct, both sides retaining screw screws.
(3) sponge pad is fixed on gum-making rack, " sandwich " is fixed on gum-making rack together with clip, add deionized water, test leakage, determine after not leaking, water outwelled and with filter paper, residual water blotted.
(4) gel of 30% and 70% is prepared respectively, wherein, the glue of 30%: 0% denaturing agent, 14mL; 100% denaturing agent, 6mL; TEMED, 10 μ L; 10% ammonium persulphate, 80 μ L.The glue of 70%: 0% denaturing agent, 6mL; 100% denaturing agent, 14mL; TEMED, 10 μ L; 10% ammonium persulphate, 80 μ L.
After (5) two syringes suck the gel of 30% and 70% respectively, discharge bubble, " lower concentration " and " high density " that enter glue according to top is fixed on gradient transfer system, and Y tube in connection, carefully drives bubble away.
(6) glass core position aimed at by syringe needle, and pin hole is towards structural glass plate, and softly also stably rotating cam transmits solution, remains a constant speed as far as possible, solution constant speed is poured in the gel slab of sandwich style.
(7) carefully insert comb, allow about hour of gel polymerisation.The equipment that rapid cleanup is finished, especially washes liquid residual in Y tube off, in order to avoid blocking.
(8) add 7L1 × TAE buffered soln (diluted by 50 × TAE buffered soln and obtain) in electrophoresis chamber, open electrophoresis control device, preheating electrophoretic buffer is to 60 DEG C.
(9) pull out away comb after polymerization, put into by glue in electrophoresis chamber, cleaning loading wells, covers temperature-control device and makes temperature rise to 60 DEG C.
3. loading: after having added LoadingBuffer in PCR primer, each glue hole adds 20 μ L.
4. electrophoresis: 80V, 12h after 120V, 30min, the principle followed be voltage (V) × time (h) close to 1000 as well.
5. dye
(1) after electrophoresis, first push one piece of sheet glass aside, then together with another block sheet glass, glue is put into pallet, glue upward.
(2) GelRed dilution: 30mL water+3 μ L10000 × GelRed mixes, lucifuge.
(3) to be divided by GelRed diluent with the pipettor of 1mL and be added on glue 3 times, each 10mL, must cover loading wells corresponding zone, add rear 10min of Denging at every turn, pallet palpus cover lid lucifuge.
(4) add deionized water and do not have glue, lucifuge 30min.
(5) rock pallet gently, make water enter between glue and sheet glass, slowly make it be separated, take out sheet glass.
6. develop: ultraviolet development under gel imaging instrument, take pictures.
The DGGE standard finger-print in 6 kinds of bifidus bacillus 16SrDNAV3 districts is as shown in Figure 3, known, and 6 kinds of bifidus bacilluss are fully separated, and is also independent of each other between the PCR primer of mixing.
(8) the DGGE qualification of unknown bifidus bacillus
1. according to embodiment 2 (two) to the operation steps of (six), obtain the PCR primer in the 16SrDNAV3 district of unknown bifidus bacillus;
2., using the hybrid standard finger printing of 6 kinds of bifidus bacilluss as Marker, the 18 strain bifidus bacilluss of the unknown are identified.
The DGGE qualification result in unknown bifidus bacillus 16SrDNAV3 district as shown in Figure 4, H as seen from the figure 1-H 3for bifidobacterium breve; H 4-H 6for the long subspecies of bifidus longum bb; H 7-H 8for bifidus longum bb baby subspecies; H 9-H 11for bifidumbacterium bifidum; H 12-H 14for animal bifidobacteria; H 15-H 18for bifidobacterium adolescentis.
The reliability demonstration of the method for Rapid Identification of Bifidobacteria of the present invention:
The result utilizing the inventive method identify the unknown bifidus bacilluss of 18 strains to obtain and 16SrDNA sequencing result compare, and the qualification result of both discoveries unanimously, thus demonstrates exactness and the reliability of this inventive method, is applicable to the strain identification of bifidus bacillus.
Specific implementation method is as follows:
1. cultivate, extract the genomic dna of bifidus bacillus according to method of the present invention;
The 16SrDNA fragment of 2.PCR amplification bifidus bacillus
(1) adopt the universal primer 27F (5 '-AGAGTTTGATCCTGGCCTCA-3 ') of bacterium and 1492R (5 ’ – GGTTACCTTGTTACGACTT-3 ') to carry out PCR, object fragment length is at about 1500bp.
(2) PCR reaction system (50 μ L) is as follows: template, 1 μ L; Upstream primer 27F (20 μMs), 0.5 μ L; Downstream primer 1492R (20 μMs), 0.5 μ L; Taq enzyme, 0.5 μ L; Taqbuffer, 5 μ L; DNTP, 5 μ L; Distilled water, 37.5 μ L.
(3) PCR response procedures: 95 DEG C, 5min; 95 DEG C, 10s; 55 DEG C, 30s; 72 DEG C, 30s; Step2-430 ×; 72 DEG C, 5min; 12 DEG C, 2min.
3. amplified production is carried out the agarose gel electrophoresis checking of concentration determination and 1%.
4. remaining PCR primer is delivered to Hua Da gene studies institute to check order.
5. sequencing result is compared with Blast on American National Biotechnology Information center (nationalcenterofbiotechnologyinformation, NCBI), determine its kind.
6. by sequencing result with compare by the result that the inventive method obtains, result is as shown in table 1, and as seen from the results in Table 1, the result that two kinds of methods obtain is completely the same in the level of planting, thus demonstrates accuracy and the reliability of this inventive method.
The sequencing result verified in table 1 the present invention compares with the qualification result using the inventive method to obtain

Claims (10)

1. a method for Rapid Identification of Bifidobacteria, is characterized in that: the method adopting PCR-DGGE, is compared by the band of unknown strains and reference culture, unknown bifidus bacillus is identified the level of kind or subspecies.
2. the method for Rapid Identification of Bifidobacteria according to claim 1, is characterized in that: step is as follows:
(1) extract the genome of the bifidus bacillus not of the same race of known sequence;
(2) adopt appropriate primer pair, carry out the V3 district of the 16SrDNA of pcr amplification list bacterium;
(3) adopt denaturing gradient gel electrophoresis method to build standard finger-print;
(4) the bifidus bacillus band of the unknown and standard band are compared, determine the kind of unknown bifidus bacillus.
3. the method for Rapid Identification of Bifidobacteria according to claim 2, is characterized in that: described step (1) in the bifidus bacillus of known sequence be bifidobacterium breve, bifidumbacterium bifidum, animal bifidobacteria, bifidobacterium adolescentis, the long subspecies of bifidus longum bb and bifidus longum bb baby subspecies.
4. the method for Rapid Identification of Bifidobacteria according to claim 2, is characterized in that: the described step (1) genomic method of middle extraction bifidus bacillus is phenol-chloroform extraction process.
5. the method for Rapid Identification of Bifidobacteria according to claim 2, is characterized in that: described step (2) middle primer pair is 338f-GC and 518r.
6. the method for Rapid Identification of Bifidobacteria according to claim 5, is characterized in that: the base sequence of described primer 338f-GC is SEQ1; The base sequence of primer 518r is SEQ2.
7. the method for Rapid Identification of Bifidobacteria according to claim 2, is characterized in that: described step (2) in the amplification condition of PCR be: 95 DEG C, 5min; 94 DEG C, 45s, 65 DEG C, 45s, each cycle down 0.5 DEG C, 72 DEG C, 30s, 20 circulations; 94 DEG C, 50s, 55 DEG C of 55s, 72 DEG C of 30s, 15 circulations; 72 DEG C of 10min.
8. the method for Rapid Identification of Bifidobacteria according to claim 2, is characterized in that: the described step glue that (3) middle denaturing gradient gel electrophoresis method is used is the polyacrylamide of 30% ~ 70%.
9. the method for Rapid Identification of Bifidobacteria according to claim 2, is characterized in that: described step (3) in the deposition condition of denaturing gradient gel electrophoresis method be: 120V, 30min; 80V, 12h; Damping fluid is 1 × TAE; Temperature is 60 DEG C.
10. the method for the Rapid Identification of Bifidobacteria according to any one of claims 1 to 3, is characterized in that: concrete steps are as follows:
(1) substratum and culture condition
Substratum: MRS+5 ‰ halfcystine, 121 DEG C of sterilizing 15min; Culture condition: 37 DEG C, the bifidus bacillus not of the same race of getting known sequence cultivates 24h in anaerobism workstation, obtains bacteria suspension;
(2) genome extracts
(1) get the bacteria suspension 1mL of overnight incubation in 1.5mL centrifuge tube, the centrifugal 2min of 10000rpm, abandons supernatant and obtains thalline;
(2), after washing thalline with the piping and druming of 1mL sterilized water, the centrifugal 2min of 10000rpm, abandons supernatant and obtains thalline;
(3) in thalline, add 200uLSDS lysate, 80 DEG C of water-bath 30min, obtain cellular lysate liquid;
(4) add phenol-chloroform solution 200uL in cellular lysate liquid, wherein the moiety of phenol-chloroform solution and volume ratio are: the saturated phenol of Tris: chloroform: primary isoamyl alcohol=25:24:1, and after putting upside down mixing, the centrifugal 5-10min of 12000rpm, gets supernatant 200uL;
(5) add 400uL ice ethanol or ice Virahol in 200uL supernatant ,-20 DEG C of centrifugal 5-10min of standing 1h, 12000rpm, abandon supernatant;
(6) add the resuspended precipitation of ice ethanol that 500uL percent by volume is 70%, the centrifugal 1-3min of 12000rpm, abandons supernatant;
(7) 60 DEG C of oven for drying, or naturally dry, obtain precipitation;
(8) 50uLddH 2the heavy molten precipitation of O, in order to PCR;
(3) 16SrDNAV3 district primer
Upstream primer 338f-GC, base sequence: SEQ1;
Downstream primer 518r, base sequence: SEQ2;
(4) PCR reaction system 50 μ L:
10 × Taqbuffer, 5 μ L; DNTP, 5 μ L; 338f-GC20 μM, 0.5 μ L; 518r20 μM, 0.5 μ L; Taq enzyme, 0.5 μ L; DNA profiling, 0.5 μ L; ddH 2o, 38 μ L;
(5) PCR response procedures
95 DEG C, 5min; 94 DEG C, 45s, 65 DEG C, each cycle down of 45s 0.5 DEG C, 72 DEG C, 30s, 20 circulations; 94 DEG C, 50s, 55 DEG C of 55s, 72 DEG C 30s15 circulation; 72 DEG C, keep 10min;
(6) agarose electrophoresis
(1) prepare the sepharose of mass percent 1%: every large glue 40mL, take 0.4g agarose and be placed in Erlenmeyer flask, add the 0.5 × TBE of 40mL, microwave-oven-heating boils to agarose and all melts, shake up, treat that solution is chilled to non-scald on hand, add 4 μ L10000 × nucleic acid dye, pour into after mixing in mould, solidify more than 30min;
(2), after genome DNA sample/PCR primer sample being mixed with 10 × Loadingbuffer, add in sample cell;
(3) switch on power, 120V, runs 30min;
(4), after electrophoresis terminates, take pictures with gel imaging instrument;
(7) operation of denaturing gradient gel electrophoresis
(1) solution preparation
1. 50 × TAE electrophoretic buffer: tris242g, Na 2eDTA2H 2o37.2g, adds 800mL deionized water, fully mixes, and adds 57.1mL Glacial acetic acid, fully mixes, and adds deionized water to 1L, mixing;
2. 0% denaturing agent 100mL: the acrylamide of mass percent 40%, 20mL; 50 × TAE electrophoretic buffer, 2mL; DH 2o, 78mL, cross 0.45 μm of filter membrane, ultrasonic degas 10-15min, is placed in brown reagent bottle;
3. 100% denaturing agent 100mL: the acrylamide of mass percent 40%, 20mL; 50 × TAE electrophoretic buffer, 2mL; Methane amide, 40mL; Urea, 42g; DH 2o, to 100mL, cross 0.45 μm of filter membrane, ultrasonic degas 10-15min, is placed in brown reagent bottle, must be heavy molten in warm water bath before using;
4. 10% ammonium persulphate: 0.1g ammonium persulphate is dissolved in 1mLdH 2in O ,-20 DEG C of storages;
(2) glue
1. the sheet glass caused not of uniform size for two panels is cleaned up, dry;
2. two spacer bars are placed in structural glass plate both sides of the edge, outward flange is mutually neat with glass plate edge, the groove of spacer bar upward, pieces of glass plate is placed on spacer bar, and align with it, and this sandwich structure clip is fixed, hold up the rear middle calibration plate that inserts and correct, both sides retaining screw screws;
3. sponge pad is fixed on gum-making rack, sandwich structure is fixed on gum-making rack together with clip, adds deionized water, test leakage, determine after not leaking, water outwelled and with filter paper, residual water blotted;
4. the gel of 30% and 70% is prepared respectively, wherein, the glue of 30%: 0% denaturing agent, 14mL; 100% denaturing agent, 6mL; Tetramethyl Ethylene Diamine, 10 μ L; 10% ammonium persulphate, 80 μ L;
The glue of 70%: 0% denaturing agent, 6mL; 100% denaturing agent, 14mL; Tetramethyl Ethylene Diamine, 10 μ L; 10% ammonium persulphate, 80 μ L;
5. after two syringes suck the gel of 30% and 70% respectively, discharge bubble, enter the lower concentration of glue and high density is fixed on gradient transfer system according to top, Y tube in connection, carefully drives bubble away;
6. glass core position aimed at by syringe needle, and pin hole is towards structural glass plate, and softly also stably rotating cam transmits solution, remains a constant speed, makes solution constant speed be poured in the gel slab of sandwich style;
7. carefully insert comb, allow gel polymerisation one hour, the equipment that rapid cleanup is finished, especially wash liquid residual in Y tube off, in order to avoid blocking;
8. add 7L1 × TAE buffered soln in electrophoresis chamber, it is diluted by 50 × TAE buffered soln and obtains, and open electrophoresis control device, preheating electrophoretic buffer is to 60 DEG C;
9. pull out away comb after polymerization, put into by glue in electrophoresis chamber, cleaning loading wells, covers temperature-control device and makes temperature rise to 60 DEG C;
(3) loading: after having added sample loading buffer in PCR primer, each glue hole adds 20 μ L;
(4) electrophoresis: 80V, 12h after 120V, 30min, the principle followed is voltage V × time h is 1000;
(5) dye
1. after electrophoresis, first push one piece of sheet glass aside, then together with another block sheet glass, glue is put into pallet, glue upward;
2. GelRed diluent: 30mL water+3 μ L10000 × GelRed mixes, and to obtain final product, to keep in Dark Place;
3. being divided by GelRed diluent with the pipettor of 1mL is added on glue 3 times, and each 10mL, must cover loading wells corresponding zone, add rear 10min of Denging at every turn, pallet palpus cover lid lucifuge;
4. add deionized water and do not have glue, lucifuge 30min;
5. rock pallet gently, make water enter between glue and sheet glass, slowly make it be separated, take out sheet glass;
(6) develop: ultraviolet development under gel imaging instrument, take pictures;
(8) the DGGE qualification of unknown bifidus bacillus
According to the operation steps of embodiment (two) to (six), obtain the PCR primer in the 16SrDNAV3 district of unknown bifidus bacillus;
Using the hybrid standard finger printing of bifidus bacillus as Marker, the bifidus bacillus of the unknown is identified.
CN201510917117.0A 2015-12-10 2015-12-10 Method for rapidly identifying bifidobacterium Pending CN105385762A (en)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107604082A (en) * 2017-09-27 2018-01-19 上海市质量监督检验技术研究院 The SNP site combination identified for bifidobacterium strain in milk powder
CN112481400A (en) * 2020-12-24 2021-03-12 山西大学 Rapid detection kit and method for food pathogenic bacteria diarrhea causing escherichia coli
CN112575098A (en) * 2020-12-24 2021-03-30 山西大学 Kit and method for rapidly detecting food pathogenic bacteria Shigella
CN112626243A (en) * 2020-12-24 2021-04-09 山西大学 Kit and method for rapidly detecting bacteria in coal geological environment
CN112921108A (en) * 2020-12-24 2021-06-08 山西医科大学 Kit and method for rapidly detecting food pathogenic bacteria salmonella
CN114778650A (en) * 2022-03-11 2022-07-22 吉林大学 Method for rapidly identifying pathogenic bacteria of blood infection by denaturing gradient gel electrophoresis

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101654705A (en) * 2009-09-09 2010-02-24 南昌大学 Application of DGGE marker of lactobacillus and bifidobacteria to flora monitoring
CN102781466A (en) * 2009-12-28 2012-11-14 芬兰血液红十字会 Probiotic bifidobacterial composition in accordance with secretor blood group status

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101654705A (en) * 2009-09-09 2010-02-24 南昌大学 Application of DGGE marker of lactobacillus and bifidobacteria to flora monitoring
CN102781466A (en) * 2009-12-28 2012-11-14 芬兰血液红十字会 Probiotic bifidobacterial composition in accordance with secretor blood group status

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NURIA SALAZAR: "Inulin-type fructans modulate intestinal Bifidobacterium species populations and decrease fecal short-chain fatty acids in obese women", 《CLINICAL NUTRITION》 *
***: "发酵乳中双岐杆菌种类快速识别", 《食品科技》 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107604082A (en) * 2017-09-27 2018-01-19 上海市质量监督检验技术研究院 The SNP site combination identified for bifidobacterium strain in milk powder
CN112481400A (en) * 2020-12-24 2021-03-12 山西大学 Rapid detection kit and method for food pathogenic bacteria diarrhea causing escherichia coli
CN112575098A (en) * 2020-12-24 2021-03-30 山西大学 Kit and method for rapidly detecting food pathogenic bacteria Shigella
CN112626243A (en) * 2020-12-24 2021-04-09 山西大学 Kit and method for rapidly detecting bacteria in coal geological environment
CN112921108A (en) * 2020-12-24 2021-06-08 山西医科大学 Kit and method for rapidly detecting food pathogenic bacteria salmonella
CN114778650A (en) * 2022-03-11 2022-07-22 吉林大学 Method for rapidly identifying pathogenic bacteria of blood infection by denaturing gradient gel electrophoresis

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