CN101712988A - Method for quickly, qualitatively and quantitatively measuring Bifidobacteria in probiotic dairy products - Google Patents

Method for quickly, qualitatively and quantitatively measuring Bifidobacteria in probiotic dairy products Download PDF

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CN101712988A
CN101712988A CN200910177909A CN200910177909A CN101712988A CN 101712988 A CN101712988 A CN 101712988A CN 200910177909 A CN200910177909 A CN 200910177909A CN 200910177909 A CN200910177909 A CN 200910177909A CN 101712988 A CN101712988 A CN 101712988A
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sample
pcr
bifidus bacillus
dna
template
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CN101712988B (en
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张和平
包秋华
孙志宏
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Inner Mongolia Agricultural University
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Inner Mongolia Agricultural University
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Abstract

The invention relates to a method for quickly, qualitatively and quantitatively measuring Bifidobacteria in probiotic dairy products. The method comprises the following steps: aiming at the Bifidobacteria in probiotic dairy products, independently designing a species specificity primer of the 16S rRNA gene sequence of the Bifidobacteria, carrying out species specificity PCR and real-time fluorescent quantitative PCR reaction by using the primer, and analyzing through an agarose gel electrophoresis pattern and a real-time fluorescent quantitative PCR pattern, thereby establishing the method for simply, conveniently, quickly, accurately, qualitatively and quantitatively measuring Bifidobacteria in probiotic dairy products.

Description

The fast qualitative of bifidus bacillus, method for quantitatively determining in the probiotic bacteria milk product
[technical field]
The present invention relates to contain the milk-product technical field of profitable probliotics.More specifically, the present invention relates to a kind of in probiotic bacteria milk product fast qualitative, the method for quantitatively determining of bifidus bacillus (Bifidobacteria).
[background technology]
Benefit lactogenesis goods become the focus of people's concern because of its abundant nutritive value and the special living effect of benefit.Probiotic bacterium and to contain the milk-product of profitable probliotics of a great variety in the market, and at full speed increase every year.To the supervision and the authentication of its quality, especially aspect qualitative and quantitative analysis, lack science, rationally, method fast.To the qualitative and quantitative analysis of probiotic bacterium, still adopt the biochemical reactions method that pure culture separates counting with selective medium in the traditional method.Traditional method is subject to factor affecting such as culture condition, strain properties, and detection efficiency is limited, and is subjected to external environment factor and substratum Effect on Performance big, detected result deficient in stability and reliability, and take time and effort.Round pcr is once fields such as molecular biology and microbiology occurring just being widely used in.The appearance of real-time fluorescence quantitative PCR (Real-time PCR) has realized the leap of round pcr from qualitative to quantitative especially, has avoided the problem of crossed contamination in the quantitative evaluation of conventional P CR.Have high specificity, good reproducibility, advantage such as accurate, quick, become the important method of molecular biology and microbiology field detection by quantitative.Adopt Bacterium lacticum kind specific PCR and real-time fluorescence quantitative PCR technology, set up easy, qualitative, the method for quantitatively determining of bifidus bacillus in the probiotic bacteria milk product fast and accurately, can simplify trace routine, the raising detection efficiency of probiotic bacterium, can improve the detectivity of China probiotic bacterium, improve the quality monitoring of protective foods, and provide technical guarantee for the long term growth of probiotic bacterium industry.
The present invention is directed to the bifidus bacillus that contains in the probiotic bacteria milk product, design the species specificity primer of the 16S rRNA gene order of bifidus bacillus voluntarily according to reference, use this primer and carry out species specificity PCR and real-time fluorescence quantitative PCR reaction, by agarose gel electrophoretogram analysis and real-time fluorescence quantitative PCR atlas analysis, set up easy, the qualitative and method for quantitatively determining fast and accurately of bifidus bacillus in the probiotic bacteria milk product.
[summary of the invention]
[technical problem that will solve]
The fast qualitative method that the purpose of this invention is to provide bifidus bacillus in a kind of probiotic bacteria milk product.
Another object of the present invention provides the fast quantification method of bifidus bacillus in a kind of probiotic bacteria milk product.
[technical scheme]
The present invention is achieved through the following technical solutions.
The present invention relates to the fast qualitative measuring method of bifidus bacillus in a kind of probiotic bacteria milk product.The step of this method is as follows:
The preparation of A, template DNA
(1) gets probiotics fermention breast sample in the 2.0mL centrifuge tube, add 500 μ L TE damping fluids, mix, place liquid nitrogen to freeze again, freeze the back and take out and put into 65 ℃ of water-baths and melt 4-6min, carry out freeze thawing 3-5 time so repeatedly;
(2) after freeze thawing finishes,, in shaking table, under 37 ℃ of temperature, shake 4h with 200r/min toward the SDS and the 10.0 μ L 10mg/mL Proteinase Ks that wherein add 60.0 μ L10% (mass volume ratio);
(3) add 100.0 μ L5M NaCl and 100 μ L CTAB/NaCl solution (4.1gNaCl is dissolved in the 80ml water, slowly adds the solution that 10g CTAB obtains again), 65 ℃ of water-bath 10min.
(4) then, add and the isopyknic phenol of solution, chloroform and the iso pentane alcohol mixture that obtain in step (3), their volume ratio is respectively 25: 24: 1, mixing, with the centrifugal 10min of 10000g/min, this solution is divided into upper strata water, middle solid phase and lower floor's organic phase again;
(5) draw the upper strata water, add with the isopyknic above-mentioned benzene chloroform of described water and iso pentane alcohol mixture extracting once, separation of supernatant;
(6) in described supernatant liquor, add the 3mol/L sodium-acetate of 0.1 times of volume and the ice Virahol of 1 times of volume, mix and leave standstill 30min, obtain total DNA precipitation with the centrifugal 5min of 10000g;
(7) described precipitation is washed 2 times with 70 volume ratio % aqueous ethanolic solutions, and seasoning at room temperature obtains template DNA again; Add the aseptic ultrapure water Hui Rong of 50.0 μ L then, standby in temperature-20 ℃ following preservation.
B, pcr amplification
Reaction system 25.0 μ L:2.5 μ L 10 * PCR Buffer, 1.0 μ L 25mmol/L Mg 2+, 2 μ L2.5mmol/L dNTPs, 2.5 μ L5mmol/L upstream and downstream primers, 1.0 μ L 50ng/ μ L dna profilings, 0.25uL 5U/ μ L Taq enzyme, redistilled water complements to 25.0 μ L;
PCR reaction conditions: 95 ℃ of pre-sex change 3min; 95 ℃ of sex change 40S, 62 ℃ of annealing 30S, 72 ℃ are extended 1min, circulate 40 times; 72 ℃ are extended 8min, obtain pcr amplification product, preserve down for 4 ℃ in temperature;
Getting 3.0 μ L PCR products uses 1% sepharose to detect;
C, result and judgement
Establish during detection to contain purpose and increase pulsating plasmid DNA as the positive contrast of template, with aqua sterilisa as the negative contrast of template; According to expection feature band occurs at the 523bp place, determine to contain bifidus bacillus in this probiotic bacteria milk product, then do not contain bifidus bacillus on the contrary.
Upstream primer Bf:5 '-GGTGGTAATGCCGGATG-3 ',
Downstream primer Br:5 '-CCACCGTTACACCGGGAA-3 ', the amplification fragment length is 523bp.
A preferred embodiment of the invention, described detection are to carry out electrophoresis 20-30min under the voltage of 5V/cm, with EB dyeing, carry out uv photography then again.
The invention still further relates to the quantitative determination method of bifidus bacillus in a kind of probiotic bacteria milk product.The step of this method is as follows:
The preparation of A, the total RNA of sample
Adopt the Trizol method to extract the total RNA of cultured milk prod, fermented sample 500mg adds 1mL Trizol reagent rapidly after grinding in filling the mortar of liquid nitrogen, presses Trizol test kit specification sheets and extracts sample RNA.Handle twice with DNaseI then, the DNA that guarantees to eliminate fully among the RNA pollutes, and obtains the purified RNA sample, uses micro-uv-spectrophotometric instrument to measure concentration, adopts 1% agarose gel electrophoresis to detect the RNA integrity, then sample is stored in-80 ℃;
B, post transcription cloning
Reaction system 10.0 μ L:2.0 μ L 5 * PrimerScript Buffer (TakaRa DRR063A), 0.5 μ L PrimerScript RT Enzyme Mix I, 0.5 μ L Random 6mers (100 μ M), total RNA (＜500ng), RNase Free dH 2O complements to 10.0 μ L;
Post transcription cloning reaction conditions: 37 ℃ of reaction 15min; 85 ℃ of sex change 5S are in temperature-20 ℃ preservation down;
C, real-time fluorescence quantitative PCR
Reaction system 25.0 μ L:12.5 μ L2 * SYBR
Figure G2009101779093D00041
Premix Ex Taq TM, each 0.5 μ L, 10 μ mol/L upstream and downstream primer Bf and Br, 2.0 μ L cDNA templates, 9.5 μ LH 2O;
Quantitative fluorescent PCR reaction parameter: 95 ℃ of sex change 25S; 95 ℃ of sex change 10S, 60 ℃ of annealing 22S, 40 circulations; In 4 ℃ of preservations of temperature; Each sample repeats 3 times;
The preparation of D, outer standard substance and the drafting of typical curve
The preparation of outer standard substance: extract and to contain with the increase DNA of pulsating plasmid pBT of Bifidobacterium animalis Subsp.Lactis V9 purpose, ultraviolet spectrophotometer calculates copy number after measuring the A value, carries out 10 times of serial dilutions, gradient dilution to 10 9-10 2The concentration of copy number/uL is carried out the quantitative fluorescent PCR reaction with this as outer standard substance;
The drafting of typical curve: the logarithm with the positive template of different copy numbers is an X-coordinate, initial cycle number (Ct) with arrival fluorescence threshold in the PCR reaction process is the typical curve that ordinate zou obtains bifidus bacillus, as the reference standard of testing sample quantitative assay;
E, result and judgement
DNA with testing sample cDNA and standard substance is a template, with the species-specific primer of bifidus bacillus, carries out the fluorescent quantitative PCR of gene fragment of the 16S rRNA of bifidus bacillus simultaneously with identical system; The fast quantification that carries out bifidus bacillus in the probiotic bacteria milk product by the Ct value of typical curve and testing sample detects.
In the present invention, described bifidus bacillus species specificity primer sequence is as follows:
Upstream primer Bf:5 '-GGTGGTAATGCCGGATG-3 ',
Downstream primer Br:5 '-CCACCGTTACACCGGGAA-3 ', the amplification fragment length is 523bp.
To explain the present invention below.
The present invention relates to the fast qualitative measuring method of bifidus bacillus in a kind of probiotic bacteria milk product.
Total DNA of total DNA of probiotic bacterium and pure culture milk-acid bacteria (comprising Bifidobacterium animalis Subsp.Lactis V9) in employing frozen-thawed-CTAB method extracting sample.Described frozen-thawed-CTAB method is the method that a kind of those skilled in the art know, and mainly is the method for utilizing the biological DNA of CTAB buffer extraction.
The step of this method is as follows:
The preparation of A, template DNA
(1) gets probiotics fermention breast sample in the 2.0mL centrifuge tube, add 500 μ L TE damping fluids, mix, place liquid nitrogen to freeze again, freeze the back and take out and put into 65 ℃ of water-baths and melt 4-6min, carry out freeze thawing 3-5 time so repeatedly.
Described TE provides a buffer environment, prevents that nucleic acid is destroyed; EDTA chelating Mg 2+Or Mn 2+Ion suppresses the DNase activity;
(2) after freeze thawing finishes, be 10% SDS and 10.0 μ L 10mg/mL Proteinase Ks, in shaking table, under 37 ℃ of temperature, shake 4h with 200r/min toward wherein adding 60.0 μ L mass volume ratios;
Described SDS is a sodium lauryl sulphate, and it is that Japanese Kanto Kagaku K. K. is with trade(brand)name SDS product sold.
Described Proteinase K is the wider serine protease of a kind of nicking activity.The carboxy-terminal peptide bond of its cutting aliphatic amino acid and die aromatischen Aminosaeuren is used for the proteinic degraded of biological sample.Purified RNA enzyme and the DNA enzymic activity removed of this kind of enzyme.Because Proteinase K is stable in urea and SDS, also has the ability of degraded natural protein, thereby it uses very extensive.The Proteinase K that the present invention uses is that AMRESCO company is with trade(brand)name Proteinase K product sold.
Described shaking table is that Shanghai ZHICHENG Anaiytical Instrument Manufacturing Co., Ltd. is with trade(brand)name ZHWY-200D constant temperature culture vibrator product sold.
(3) add 100.0 μ L 5M NaCl and 100 μ L 10%CTAB solution (4.1gNaCl is dissolved in and slowly adds CTAB10g in the 80ml water), 65 ℃ of water-bath 10min.
Described NaCl provides a salt environment, DNA is fully dissolved and is present in the liquid phase.
Described CTAB is a cetyl trimethylammonium bromide, it is a kind of cationic detergent, characteristic with precipitate nucleic acids and acidic polysaccharide from the solution of low ionic strength, under this condition, protein and neutral saccharan are still stayed in the solution, in the solution of high ionic strength, the saccharan beyond CTAB and protein and the most of acidic polysaccharide forms mixture, just can not precipitate nucleic acids.At last by ethanol or isopropanol precipitating DNA, and CTAB is dissolved in ethanol or Virahol and remove.
(4) then, add and the isopyknic phenol of solution, chloroform and the iso pentane alcohol mixture that obtain in step (3), their volume ratio is respectively 25: 24: 1, mixing, with the centrifugal 10min of 10000g/min, this solution is divided into upper strata water, middle solid phase and lower floor's organic phase again.
(5) draw the upper strata water, add with the isopyknic above-mentioned phenol of described water, chloroform and iso pentane alcohol mixture extracting once, separation of supernatant.
(6) in described supernatant liquor, add the 3mol/L sodium-acetate of 0.1 times of volume and the ice Virahol of 1 times of volume, mix, leave standstill 30min again,, obtain total DNA precipitation with the centrifugal 5min of 10000g;
(7) described precipitation is washed 2 times with 70 volume % aqueous ethanolic solutions, and seasoning at room temperature obtains template DNA again; Add the aseptic ultrapure water Hui Rong of 50 μ L then, standby in temperature-20 ℃ following preservation.
B, pcr amplification
Pcr amplification is a kind of technology of external efficient repetition DNA, and this technology is almost through present molecular biological every field.The PCR system is made up of template, Auele Specific Primer, pyro polymerase, several parts of thymus nucleic acid.The pcr amplification process is can the branch template high-temperature denatured, primer and template in low temperature annealing, primer extend under the effect of pyro polymerase.
This reaction system 25.0 μ L:2.5 μ L10 * PCR Buffer, 1.0 μ L 25mmol/L Mg 2+, 2 μ L2.5mmol/L dNTPs, 2.5 μ L 5mmol/L upstream and downstream primers, 1.0 μ L 50ng/ μ LDNA templates, 0.25uL 5U/ μ L Taq enzyme, redistilled water complements to 25.0 μ L.
In the present invention, described upstream and downstream primer is respectively:
Upstream primer Bf:5 '-GGTGGTAATGCCGGATG-3 ',
Downstream primer Br:5 '-CCACCGTTACACCGGGAA-3 ', the amplification fragment length is 523bp.
PCR reaction conditions: 95 ℃ of pre-sex change 3min; 95 ℃ of sex change 40S, 62 ℃ of annealing 30S, 72 ℃ are extended 1min, circulate 40 times; 72 ℃ are extended 8min, obtain pcr amplification product, preserve down for 4 ℃ in temperature;
Getting 3.0 μ LPCR products uses 1% sepharose to detect.The instrument that the agarose gel electrophoretogram analysis is to use UVP company to sell with trade(brand)name CDS-8000 gel imaging instrument carries out.
C, result and judgement
Establish during detection to contain purpose and increase pulsating plasmid DNA as the positive control template, with aqua sterilisa as the negative control template; According to expection feature band occurs at the 523bp place, determine to contain bifidus bacillus in this probiotic bacteria milk product, then do not contain bifidus bacillus on the contrary.Specifically can be referring to attached Fig. 1 and 2.
The invention still further relates to the quantitative determination method of bifidus bacillus in a kind of probiotic bacteria milk product.The step of this method is as follows:
The preparation of A, the total RNA of sample
Adopt the Trizol method to extract the total RNA of cultured milk prod, fermented sample 500mg adds 1mL Trizol reagent rapidly after grinding in filling the mortar of liquid nitrogen, presses Trizol reagent specification sheets and extracts sample RNA.Handle twice with DNaseI then, the DNA that guarantees to eliminate fully among the RNA pollutes, and obtains the purified RNA sample, measures concentration, in temperature-80 ℃ preservation down;
Described Trizol reagent is to specialize in the product that extracts RNA by Invitrogen company; DNase I is a TaKaRa company product; Reagent such as the chloroform that uses in this process, Virahol, dehydrated alcohol are the products that chemical reagent three factories in Tianjin produce.
RNA has maximum absorption peak at 260nm wavelength place.Therefore, use DEPC-dH 2O dissolved RNA sample can directly use the trace of NanoDrop-1000 for example ultraviolet spectrophotometer to be determined at the RNA concentration and the purity at 260nm wavelength place, DEPC-H 2O is a blank, and RNA concentration can direct reading, and RNA purity is passed through OD 260/280Detect.If ratio between 1.9-2.1, thinks that purity is better, ratio has heteroproteins more less than 1.8 explanations; Ratio shows then that greater than 2.2 RNA degrades.
The RNA integrity is by 1% agarose gel electrophoresis.
B, post transcription cloning
Reaction system 10.0 μ L:2.0 μ L5 * PrimerScript Buffer (TakaRa DRR063A), 0.5 μ LPrimerScript RT Enzyme Mix I, 0.5 μ L Random 6mers (100 μ M), total RNA (＜500ng), RNase Free dH 2O complements to 10.0 μ L;
Post transcription cloning reaction conditions: 37 ℃ of reaction 15min; 85 ℃ of sex change 5S are in temperature-20 ℃ preservation down;
C, real-time fluorescence quantitative PCR
Reaction system 25.0 μ L:12.5 μ L2 * SYBR
Figure G2009101779093D00071
Premix Ex TaqTM, each 0.5 μ L10 μ mol/L upstream and downstream primer Bf and Br, 2.0 μ L cDNA templates, 9.5 μ LdH2O;
Quantitative fluorescent PCR reaction parameter: 95 ℃ of sex change 25S; 95 ℃ of sex change 10S, 60 ℃ of annealing 22S, 40 circulations; In 4 ℃ of preservations of temperature; Each sample repeats 3 times;
The preparation of D, outer standard substance and the drafting of typical curve
The preparation of outer standard substance: extract and to contain with the bifidus bacillus Bifidobacterium animalis Subsp.Lactis V9 purpose pulsating plasmid DNA that increases, after ultraviolet spectrophotometer is measured the A value, calculate copy number, carry out 10 times of serial dilutions, gradient dilution to 10 9-10 2The concentration of copy number/uL is carried out the quantitative fluorescent PCR reaction with this as outer standard substance;
The drafting of typical curve: outer standard substance are carried out 10 times of serial dilutions, make it become the template of the certain gradient copy number of tool content, logarithm with the positive template of different copy numbers is an X-coordinate, initial cycle number (Ct) with arrival fluorescence threshold in the PCR reaction process is the typical curve (referring to accompanying drawing 3) that ordinate zou obtains bifidus bacillus, for the testing sample quantitative assay provides reference standard;
E, result and judgement
With the cDNA of testing sample and the DNA of standard substance is template, with the species-specific primer of bifidus bacillus, carries out the fluorescent quantitative PCR of gene fragment of the 16S rRNA of bifidus bacillus simultaneously with identical system; The fast quantification that carries out bifidus bacillus in the probiotic bacteria milk product by the Ct value of typical curve and testing sample detects.
The real-time fluorescence quantitative PCR atlas analysis is to use Bio-Red company with trade(brand)name iQ TM5 instruments of selling carry out.
In the present invention, described bifidus bacillus species specificity primer sequence is as follows:
Upstream primer Bf:5 '-GGTGGTAATGCCGGATG-3 ',
Downstream primer Br:5 '-CCACCGTTACACCGGGAA-3 ', the amplification fragment length is 523bp.
The errot analysis of bifidus bacillus quantitative determination method is as follows in the probiotic bacteria milk product of the present invention: compare with traditional PCR, quantitative PCR is quicker, sensitive, and can reduce the crossed contamination that produces in the experimentation effectively.Because quantitative PCR has high specificity, susceptibility height, advantage that accuracy is good, so it operates the poor reliability that each process all may cause the quantitative PCR experiment result, as: the application of sample of sample collection and storage, extraction RNA, quantitative PCR detection process etc., so when using quantitative PCR technique, the stdn that is applied to test, rationalization, detailed-oriented and effectuation make quantitative PCR technique be our service better.
The preparation of standard substance is key links of setting up real-time fluorescence quantitative PCR, and standard substance commonly used have three kinds of construction processs: the one, according to one section oligonucleotide of amplified fragments sequence synthetic; The 2nd, reclaim purifying and contain the segmental conventional pcr amplification product of fluorescent quantitative PCR; The 3rd, in plasmid, reclaim plasmid with containing the fluorescent quantitative PCR fragment cloning.The present invention selects the third method, made up contain the purpose band recombinant plasmid as standard substance, as standard substance production standard curve the absolute magnitude (copy number) of unknown sample is measured.
[beneficial effect]
The invention has the beneficial effects as follows:
Can not carry out on the basis of probiotic bacterium pure culture, easy, the bifidus bacillus in the qualitative and quantitative analysis probiotic bacteria milk product quickly and accurately, whole process is simple to the trace routine of bifidus bacillus in the probiotic bacteria milk product, detection efficiency is high, accuracy good.
[description of drawings]
The primer specificity checking of Fig. 1, bifidus bacillus
M:DL2000 Marker; 1.pBT as positive control; 2.dH 2O makes negative control; 3.Bifidobacterium animalis Subsp.Lactis V9; 4.L.plantarum 1.2437; 5.L.rhamnosus 1.2134; 6.L.coryniformis subsp.coryniformis 1.1879.7.L.caseiATCC393,8.L.acidophilus ATCC4356; 9.L.Reuteri ZL12-1-1
Bifidus bacillus qualitative detection in Fig. 2, the milk-product
1, sour milk sample 12# (this experiment L.casei Zhang and Bifidobacterium animalis Subsp.Lactis V9 mixed fermentation sample); 2.H 2O makes negative control; 3. sour milk sample 25#; (the traditional zymotic breast of adopting from Tibet); 4, sour milk sample 36# (the spontaneous fermentation breast of adopting from Tibet)
The typical curve of Fig. 3, bifidus bacillus.
The melting point curve of Fig. 4 bifidus bacillus.
[embodiment]
The invention will be further described below in conjunction with embodiment.
Embodiment 1
The fast qualitative of bifidus bacillus detects in the probiotic bacteria milk product:
This embodiment use contains purpose and increases pulsating plasmid pBT as positive control; DH 2O makes negative control; Use Bifidobacterium animalis Subsp.Lactis V9, L.plantarum1.2437, L.rhamnosus 1.2134, L.coryniformis subsp.coryniformis 1.1879, L.casei ATCC393, L.acidophilus ATCC4356, L.reuteri ZL12-1-1 in addition.And sour milk sample 12# (L.casei Zhang and Bifidobacterium animalis Subsp.Lactis V9 mixed fermentation), the traditional zymotic yoghourt sample 25# that adopts from Tibet, the spontaneous fermentation yoghourt sample 36# that adopts from Tibet carry out as sample.See Fig. 1 and 2.
Described L.rhamnosus 1.2134, L.plantarum 1.2437, L.coryniformis subsp.coryniformis 1.1879 are available from Chinese common micro-organisms DSMZ; L.acidophilus ATCC4356, L.casei ATCC393 are available from U.S. American Type Culture Collection.Bifidobacterium animalis Subsp.Lactis V9, L.reuteri ZL 12-1-1 are the preservation of key lab of the Ministry of Education of Agricultural University of the Inner Mongol's " dairy products biotechnology and engineering ".
A. the preparation of template DNA
Adopt frozen-thawed-CTAB method extracting sample DNA, concrete steps are as follows:
(1) gets the milk-acid bacteria (comprising Bifidobacterium animalis Subsp.Lactis V9) of 0.5g probiotics fermention breast sample or MRS culture medium culturing in the 2.0mL centrifuge tube, add 500 μ L TE damping fluids, mix, place liquid nitrogen to freeze again, freeze the back and take out and to put into 65 ℃ of water-baths and melt 5min, carry out freeze thawing so repeatedly 4 times;
(2) after freeze thawing finishes,, in shaking table, under 37 ℃ of temperature, shake 4h with 200r/min toward the SDS and the 10.0 μ L 10mg/mL Proteinase Ks that wherein add 60.0 μ L10% (mass volume ratio);
(3) add 100.0 μ L 5M NaCl and 100 μ L CTAB/NaCl (4.1gNaCl is dissolved in the 80ml water, slowly adds the solution that 10g CTAB obtains again), 65 ℃ of water-bath 10min.
(4) then, add and the isopyknic phenol of solution, chloroform and the iso pentane alcohol mixture that obtain in step (3), their volume ratio is respectively 25: 24: 1, mixing, with the centrifugal 10min of 10000g/min, this solution is divided into upper strata water, middle solid phase and lower floor's organic phase again;
(5) draw the upper strata water, add with the isopyknic above-mentioned benzene chloroform of described water and iso pentane alcohol mixture extracting once, separation of supernatant;
(6) in described supernatant liquor, add the 3mol/L sodium-acetate of 0.1 times of volume and the ice Virahol of 1 times of volume, mix and leave standstill 30min, obtain total DNA precipitation with the centrifugal 5min of 10000g;
(7) described precipitation is washed 2 times with 70 volume % aqueous ethanolic solutions, and seasoning at room temperature obtains template DNA again; Add the aseptic ultrapure water Hui Rong of 50.0 μ L then, standby in temperature-20 ℃ following preservation.
B.PCR detects
Reaction system 25.0 μ L:2.5 μ L 10 * PCR Buffer (contains Mg 2+), 2 μ L 2.5mmol/LdNTPs, totally 2.5 μ L 5mmol/L upstream and downstream primers (Bf and Br), 1.0 μ L 50ng/ μ L dna profilings, 0.25 μ L 5U/ μ L Taq enzyme (TakaRa) complements to 25.0 μ L with redistilled water.With PCR product voltage with 5V/cm in 1% sepharose, electrophoresis 20-30min, EB dyeing, ultraviolet is taken pictures.
C. the PCR product with Bifidobacterium animalis Subsp.Lactis V9 is connected conversion DH5 α with pMD 18, obtains the sub-pBT of positive colony.
D. result and judgement
Through the amplification of the species specificity primer PCR of bifidus bacillus, the agarose gel electrophoresis assay is seen Fig. 1, and swimming lane 1 increases pulsating plasmid pBT as positive control to contain purpose, swimming lane 2 with aqua sterilisa as negative control.Swimming lane 3 is preserved bacterial strain Bifidobacterium animalis Subsp.Lactis V9 for the laboratory; Swimming lane 4-9 is outer other kinds milk-acid bacteria of Bifidobacteria, and the result is negative, illustrates that primer specificity is good, and is consistent with expected results.
The qualitative detection of milk-product sample is seen Fig. 2, it is generally acknowledged that the expection amplified band appears in positive feature at the 523bp place, illustrate in this sample and contain bifidus; Negative control does not have the feature band.Sample 12# is the sour milk that utilizes this breadboard 2 probiotics (L.casei Zhang and Bifidobacterium animalis Subsp.Lactis V9) mixed fermentation to make in the swimming lane 1, wherein contain bifidus bacillus, the result is consistent with expected results, has positive feature; Spontaneous fermentation breast 25# and the 36# sample of swimming lane 3-4 for adopting from Tibet, the result is negative, illustrates that this sample does not contain Bifidobacterium.
Embodiment 2.
The quantitative assay of bifidus bacillus in the probiotic bacteria milk product:
The different samples of mentioning among the embodiment 1 have been carried out the quantitative assay of bifidus bacillus.
This embodiment uses sour milk sample 12# (L.casei Zhang and Bifidobacterium animalisSubsp.Lactis V9 mixed fermentation) and commercialization bacterial strain BL12 to carry out the quantitative assay of bifidus bacillus, and its determination step is as follows:
A. the preparation of the total RNA of sample
Adopt the Trizol method to extract: after sample 500mg grinds in filling the mortar of liquid nitrogen, add 1mL Trizol reagent rapidly, press Trizol test kit specification sheets and extract sample RNA, handle twice with DNaseI then, the DNA that guarantees to eliminate fully among the RNA pollutes, obtain the purified RNA sample, carry out concentration determination according to the method for present specification explanation, the RNA integrity is by 1% agarose gel electrophoresis.
B. post transcription cloning
Reaction system 10.0 μ L:2.0 μ L 5 * PrimerScript Buffer (TakaRa DRR063A), 0.5 μ LPrimerScript RT Enzyme Mix I, 0.5 μ L Random 6mers (100 μ M), total RNA (＜500ng), RNase Free dH 2O complements to 10.0 μ L;
Post transcription cloning reaction conditions: 37 ℃ of reaction 15min; 85 ℃ of sex change 5S are in temperature-20 ℃ preservation down;
C. real-time fluorescence quantitative PCR
Quantitative fluorescent PCR is according to SYBR
Figure G2009101779093D00121
PrimeScript TMRT-PCR Kit (the precious biotechnology in Dalian company limited, TaKaRa DRR063A) specification sheets carries out.Reaction system 25.0 μ L:12.5 μ LSYBR Premix Ex Taq TM(2 *), each 0.5 μ L, 10 μ mol/L upstream and downstream primer (Bf and Br), 2.0 μ L cDNA templates, 9.5 μ L redistilled waters.Quantitative fluorescent PCR reaction parameter: 95 ℃ of sex change 25S; 95 ℃ of sex change 5S, 60 ℃ of annealing 22S, 40 circulations; In 4 ℃ of preservations of temperature.
D. the drafting of the preparation of outer standard substance and typical curve
Extraction is to contain the purpose total DNA of pulsating plasmid pBT that increases, and ultraviolet spectrophotometer calculates copy number after measuring the A value, does 10 times of serial dilutions, gradient dilution to 10 9-10 2The concentration of copy number/uL is carried out the quantitative fluorescent PCR reaction with this as outer standard substance.Logarithm with the positive template of different copy numbers is an X-coordinate, is the typical curve that ordinate zou obtains bifidus bacillus with the initial cycle number (Ct) that arrives fluorescence threshold in the PCR reaction process, sees accompanying drawing 3.
F, result and analysis
As seen from Figure 3, Ct value and copy number are the better linearity relation, (Y represents the Ct value to quantitative fluorescent PCR typical curve equation: Y=-3.267X+42, x represents the logarithm of original template amount), linearly dependent coefficient is 0.994 between original template concentration and the Ct value, slope is-3.267, and the bifidus bacillus typical curve of being set up meets the quantitative requirement of Real-Time PCR.As seen from Figure 4, the melting point curve unanimity of sample amplification product, and be the curve of simple spike type all, proving that the quantitative reaction systemic characteristic of fluorescence dye is good, primer and PCR reflection condition all are fit to carry out fluorescent quantitation.
CDNA and standard substance DNA with testing sample are template, and the species-specific primer of two qi Bacterium lacticum of describing with embodiment 1 carries out the fluorescent quantitative PCR of gene fragment of the 16S rRNA of pair qi Bacterium lacticum simultaneously with identical system.IQ by typical curve and employing Bio-Red company TMThe fast quantification that the computer software analysis of 5 multiple real time fluorescence quantifying PCR instrument carries out two qi Bacterium lacticum in the probiotic bacteria milk product detects.The measurement result of these samples and analytical error result are listed in the table below in 1 in the lump.
The Real-time measurement result of table 1 Bifidobacteria fermented-milk sample
Sample X+SD copy number/g fermented-milk
L.casei Zhang and Bifidobacteriumanimalis Subsp.Lactis V9 mixing cultured milk ??8.84±0.5
??Bifidobacteriumaninalis?Bb12 ??8.54±0.5
Result by table 1 also clearly illustrates that, quantitative PCR can be accurately quantitative living bifidobacteria in the fermented-milk.
Sequence table
＜110〉Agricultural University of the Inner Mongol
＜120〉fast qualitative of bifidus bacillus, method for quantitatively determining in the probiotic bacteria milk product
<130>
<160>2
<210>1
<211>17
<212>DNA
＜213〉artificial sequence
<220>
＜223〉to the description of artificial sequence: the designed and upstream primer Bf that uses of the present invention.
<400>1
GGTGGTAATG?CCGGATG????17
<210>2
<211>18
<212>DNA
＜213〉artificial sequence
<220>
＜223〉to the description of artificial sequence: the designed and downstream primer Br that uses of the present invention.
<400>2
CCACCGTTAC?ACCGGGAA????18

Claims (5)

1. the fast qualitative measuring method of bifidus bacillus in the probiotic bacteria milk product is characterized in that the step of this method is as follows:
The preparation of A, template DNA
(1) gets probiotics fermention breast sample in the 2.0mL centrifuge tube, add 500.0 μ L TE damping fluids, mix, place liquid nitrogen to freeze fully again, freeze the back and take out and put into 65 ℃ of water-baths and melt 4-6min, carry out freeze thawing 3-5 time so repeatedly;
(2) after freeze thawing finishes, toward wherein adding 60.0 μ L 10% (mass volume ratio) SDS and 10.0uL
The 10mg/mL Proteinase K shakes 4h with 200r/min under 37 ℃ of temperature in shaking table;
(3) add 100.0 μ L 5M NaCl and 100.0 μ L10%CTAB/NaCl solution, 65 ℃ of water-bath 10min;
(4) add and the isopyknic phenol of solution, chloroform and the iso pentane alcohol mixture that obtain in step (3), their volume ratio is respectively 25: 24: 1, mixing, again with the centrifugal 10min of 10000g/min, this solution is divided into upper strata water, middle solid phase and lower floor's organic phase;
(5) draw the upper strata water, add with the isopyknic above-mentioned phenol of described water, chloroform and iso pentane alcohol mixture extracting once, separation of supernatant;
(6) in described supernatant liquor, add the 3mol/L sodium-acetate of 0.1 times of volume and the ice Virahol of 1 times of volume, mix, leave standstill 30min again,, obtain total DNA precipitation with the centrifugal 5min of 10000g/min;
(7) described precipitation is washed 2 times with 70% (volume ratio) aqueous ethanolic solution, and seasoning at room temperature obtains template DNA again; Add the aseptic ultrapure water Hui Rong of 50.0 μ L then, standby in temperature-20 ℃ following preservation;
B, pcr amplification
Reaction system 25.0 μ L:2.5 μ L 10 * PCR Buffer, 1.0 μ L 25mmol/L Mg 2+, 2.0 μ L2.5mmol/L dNTPs, 2.5 μ L 5mmol/L upstream and downstream primers, 1.0 μ L 50ng/ μ LDNA templates, 0.25uL 5U/ μ L Taq enzyme, redistilled water complements to 25.0 μ L;
PCR reaction conditions: 95 ℃ of pre-sex change 3min; 95 ℃ of sex change 40S, 62 ℃ of annealing 30S, 72 ℃ are extended 1min, circulate 40 times; 72 ℃ are extended 8min, obtain pcr amplification product, preserve down for 4 ℃ in temperature;
Getting 3.0 μ L PCR products uses 1% sepharose to detect;
C, result and judgement
Establish during detection to contain purpose and increase pulsating plasmid DNA as the positive contrast of template, with aqua sterilisa as the negative contrast of template; According to expection feature band occurs at the 523bp place, determine to contain bifidus bacillus in this probiotic bacteria milk product, then do not contain bifidus bacillus on the contrary.
2. method according to claim 1 is characterized in that described upstream and downstream primer is respectively:
Upstream primer Bf:5 '-GGTGGTAATGCCGGATG-3 ',
Downstream primer Br:5 '-CCACCGTTACACCGGGAA-3 ', the amplification fragment length is 523bp.
3. method according to claim 1, it is characterized in that containing the purpose pulsating plasmid DNA that increases is to be connected positive colony that transformed into escherichia coli DH5 α obtains with pMD18 by the PCR product with Bifidobacterium animalis Subsp.Lactis V9.
4. the quantitative determination method of bifidus bacillus (Bifidobacteria) in the probiotic bacteria milk product is characterized in that the step of this method is as follows:
The preparation of A, the total RNA of sample
Adopt the Trizol method to extract the total RNA of cultured milk prod: after fermented sample 500mg grinds in filling the mortar of liquid nitrogen, add 1mL Trizol reagent rapidly, press Trizol test kit specification sheets and extract sample RNA, handle twice with DNaseI then, the DNA that guarantees to eliminate fully among the RNA pollutes, and obtains the purified RNA sample, uses micro-uv-spectrophotometric instrument to measure concentration, adopt 1% agarose gel electrophoresis to detect the RNA integrity, then sample is stored in-80 ℃;
B, post transcription cloning
Reaction system 10.0 μ L:2.0 μ L 5 * PrimerScript Buffer (TakaRa DRR063A), 0.5 μ L PrimerScript RT Enzyme Mix I, 0.5 μ L Random 6mers (100 μ M), TotalRNA (＜500ng), RNase Free dH 2O complements to 10.0 μ L;
Post transcription cloning reaction conditions: 37 ℃ of reaction 15min; 85 ℃ of sex change 5S are in temperature-20 ℃ preservation down;
C, real-time fluorescence quantitative PCR
Reaction system 25.0 μ L:12.5 μ L
Figure F2009101779093C00021
Ex Taq TM, each 0.5 μ L, 10 μ mol/L upstream and downstream primer Bf and Br, 2.0 μ L cDNA templates, 9.5 μ LdH 2O;
Quantitative fluorescent PCR reaction parameter: 95 ℃ of sex change 25S; 95 ℃ of sex change 10S, 60 ℃ of annealing 22S, 40 circulations; Each sample repeats 3 times; In 4 ℃ of preservations of temperature;
The preparation of D, outer standard substance and the drafting of typical curve
The preparation of outer standard substance: extract and to contain with the increase DNA of pulsating plasmid pBT of bifidus bacillus Bifidobacterium animalis Subsp.Lactis V9 purpose, after ultraviolet spectrophotometer is measured the A value, calculate copy number, carry out 10 times of serial dilutions, gradient dilution to 10 9-10 2The concentration of copy number/μ L is carried out the quantitative fluorescent PCR reaction with this as outer standard substance;
The drafting of typical curve: the logarithm with the positive template of different copy numbers is an X-coordinate, initial cycle number (Ct) with arrival fluorescence threshold in the PCR reaction process is the typical curve that ordinate zou obtains bifidus bacillus, as the reference standard of testing sample quantitative assay;
E, result and judgement
DNA with testing sample cDNA and standard substance is a template, with the species-specific primer of bifidus bacillus, carries out the fluorescent quantitative PCR of gene fragment of the 16S rRNA of bifidus bacillus simultaneously with identical system; Obtain the starting template amount of bifidus bacillus in the probiotic bacteria milk product by the Ct value of typical curve and testing sample.
5. method according to claim 4 is characterized in that described bifidus bacillus species specificity primer sequence is as follows:
Upstream primer Bf:5 '-GGTGGTAATGCCGGATG-3 ',
Downstream primer Br:5 '-CCACCGTTACACCGGGAA-3 ', the amplification fragment length is 523bp.
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