CN101712986B - Method for quickly, qualitatively and quantitatively measuring Lactobacillus acidophilus in probiotic dairy products - Google Patents

Abstract

The invention relates to a method for quickly, qualitatively and quantitatively measuring Lactobacillus acidophilus in probiotic dairy products. The method comprises the following steps: aiming at the Lactobacillus acidophilus in probiotic dairy products, independently designing a species specificity primer of the 16S rRNA gene sequence of the Lactobacillus acidophilus, 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 Lactobacillus acidophilus in probiotic dairy products.

Description

The fast qualitative of Lactobacterium acidophilum, 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 Lactobacterium acidophilum (Lactobacillus acidophilus).

[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 of scientific, 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 receives external environment factor and substratum Effect on Performance big, detected result deficient in stability and safety, 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 probiotic lactobacillus kind specific PCR and real-time fluorescence quantitative PCR technology; Set up easy, qualitative, the method for quantitatively determining of Lactobacterium acidophilum 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 technical guarantee is provided for the long term growth of probiotic bacterium industry.

The present invention is directed to the Lactobacterium acidophilum that contains in the probiotic bacteria milk product; Design the species specificity primer of the 16S rRNA gene order of Lactobacterium acidophilum voluntarily according to reference; Use this primer and carry out species specificity PCR and real-time fluorescence quantitative PCR reaction; Through 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 Lactobacterium acidophilum 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 Lactobacterium acidophilum in a kind of probiotic bacteria milk product.

Another object of the present invention provides the fast quantification method of Lactobacterium acidophilum in a kind of probiotic bacteria milk product.

[technical scheme]

The present invention realizes through following technical proposals.

The present invention relates to the fast qualitative measuring method of Lactobacterium acidophilum in a kind of probiotic bacteria milk product.The step of this method is following:

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 L 10% (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 said water and iso pentane alcohol mixture extracting once, separation of supernatant;

(6) in described supernatant, 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 deposition with the centrifugal 5min of 10000g/min;

(7) said deposition is with 70 volume ratio % aqueous ethanolic solutions washing 2 times, and seasoning at room temperature obtains template DNA again; Add the aseptic ultrapure water Hui Rong of 50.0 μ L then, subsequent use 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 L 5mmol/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 preparatory sex change 3min; 95 ℃ of sex change 40S, 57 ℃ 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 DNA as the positive contrast of template, with aqua sterilisa as the negative contrast of template; According to expection characteristic band occurs at the 759bp place, confirm to contain Lactobacterium acidophilum in this probiotic bacteria milk product, then do not contain Lactobacterium acidophilum on the contrary.

Upstream primer Af is 5 '-AGCTGAACCAACAGATTCAC-3 ',

Downstream primer Ar is 5 '-ACTACCAGGGTATCTAATCC-3 ', and the amplification fragment length is 759bp.

A preferred embodiment of the invention, described detection are under the voltage of 5V/cm, to carry out electrophoresis 20-30min, with EB dyeing, carry out uv photography then again.

The invention still further relates to the quantitative determination method of Lactobacterium acidophilum in a kind of probiotic bacteria milk product.The step of this method is following:

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 in filling the mortar of liquid nitrogen, grinding, and 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 appearance 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

Premix Ex Taq ^TM, each 0.5 μ L10 μ mol/L upstream and downstream primer Af and Ar, 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, 56 ℃ 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 pAT of Lactobacterium acidophilum ATCC4356 (available from U.S. American Type Culture Collection) 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/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) to arrive fluorescence threshold in the PCR reaction process is the typical curve that ordinate zou obtains Lactobacterium acidophilum, as the reference standard of testing sample quantitatively determined;

E, result and judgement

DNA with testing sample cDNA and standard substance is a template, with the species-specific primer of Lactobacterium acidophilum, carries out the fluorescent quantitative PCR of gene fragment of the 16S rRNA of Lactobacterium acidophilum simultaneously with identical system; The fast quantification that carries out Lactobacterium acidophilum in the probiotic bacteria milk product through the Ct value of typical curve and testing sample detects.

In the present invention, said Lactobacterium acidophilum species specificity primer sequence is following:

Upstream primer Af is 5 '-AGCTGAACCAACAGATTCAC-3 ',

Downstream primer Ar is 5 '-ACTACCAGGGTATCTAATCC-3 ', and the amplification fragment length is 759bp.

Below the present invention will be described at length.

The present invention relates to the fast qualitative measuring method of Lactobacterium acidophilum in a kind of probiotic bacteria milk product.

Total DNA of total DNA of probiotic bacterium and pure culture milk-acid bacteria (comprising Lactobacillus acidophilus ATCC4356) in employing frozen-thawed-CTAB method extracting sample.Described frozen-thawed-CTAB method is the method that the technician in a kind of present technique field knows, and mainly is the method for utilizing the biological DNA of CTAB buffer extraction.

The step of this method is following:

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, it is active to suppress DNase;

(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 Tryase 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 the 80ml water, slowly adds the solution that 10g CTAB obtains again), 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; Be a kind of cationic detergent, have the characteristic of precipitate nucleic acids and acidic polysaccharide from the solution of LIS, 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 through 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 said water, chloroform and iso pentane alcohol mixture extracting once, separation of supernatant.

(6) in described supernatant, 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 deposition with the centrifugal 5min of 10000g;

(7) said deposition is with 70 volume % aqueous ethanolic solutions washing 2 times, and seasoning at room temperature obtains template DNA again; Add the aseptic ultrapure water Hui Rong of 50 μ L then, subsequent use in temperature-20 ℃ following preservation.

B, pcr amplification

Pcr amplification is the technology of a kind of external efficient replication DNA, and this technique almost is 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 below in the effect of pyro polymerase.

This 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 L 5mmol/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.

In the present invention, described upstream and downstream primer is respectively:

Upstream primer Af is 5 '-AGCTGAACCAACAGATTCAC-3 ',

Downstream primer Ar is 5 '-ACTACCAGGGTATCTAATCC-3 ', and the amplification fragment length is 759bp.

PCR reaction conditions: 95 ℃ of preparatory sex change 3min; 95 ℃ of sex change 40S, 57 ℃ 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 appearance carries out.

C, result and judgement

Establish during detection to contain purpose and increase pulsating DNA as the positive control template, with aqua sterilisa as the negative control template; According to expection characteristic band occurs at the 759bp place, confirm to contain Lactobacterium acidophilum in this probiotic bacteria milk product, then do not contain Lactobacterium acidophilum on the contrary.Specifically can be referring to accompanying drawing 1.

The invention still further relates to the quantitative determination method of Lactobacterium acidophilum in a kind of probiotic bacteria milk product.The step of this method is following:

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 in filling the mortar of liquid nitrogen, grinding, and 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 the TaKaRa Company products; Reagent such as the chloroform that uses in this process, Virahol, absolute ethyl alcohol are the products that chemical reagent three factories in Tianjin produce.

RNA has maximum absorption peak in the 260nm wavelength.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 of 260nm wavelength, 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 detects through 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 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 L 2 * SYBR

Premix Ex TaqTM, each 0.5 μ L10 μ mol/L upstream and downstream primer Af and Ar, 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, 56 ℃ 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 Lactobacillus acidophilus ATCC4356 purpose pulsating DNA that increases, 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: 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) to arrive fluorescence threshold in the PCR reaction process is the typical curve (referring to accompanying drawing 2) that ordinate zou obtains Lactobacterium acidophilum, for the testing sample quantitatively determined 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 Lactobacterium acidophilum, carries out the fluorescent quantitative PCR of gene fragment of the 16S rRNA of Lactobacterium acidophilum simultaneously with identical system; The fast quantification that carries out Lactobacterium acidophilum in the probiotic bacteria milk product through 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 ^TMThe instrument of 5 sale carries out.

In the present invention, said Lactobacterium acidophilum species specificity primer sequence is following:

Upstream primer Af is 5 '-AGCTGAACCAACAGATTCAC-3 ',

Downstream primer Ar is 5 '-ACTACCAGGGTATCTAATCC-3 ', and the amplification fragment length is 759bp.

The errot analysis of Lactobacterium acidophilum quantitative determination method is following in the probiotic bacteria milk product of the present invention:

CR compares with traditional P, and quantitative PCR is quicker, sensitive, and can reduce the crossed contamination that produces in the experimentation effectively.Because quantitative PCR has the advantage that high specificity, susceptibility are high, particularity is good; So its each process of operation all possibly cause quantitative PCR experiment result's poor reliability; 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, carefulization 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 Lactobacterium acidophilum in the qualitative and quantitative analysis probiotic bacteria milk product quickly and accurately, whole process is simple to the trace routine of Lactobacterium acidophilum in the probiotic bacteria milk product, detection efficiency is high, accuracy good.

[description of drawings]

The primer specificity checking of Fig. 1, Lactobacterium acidophilum and the qualitative detection of sample

M:DL2000Marker; 1.pAT as positive control; 2.dH ₂O makes negative control; 3.L.acidophilus ATCC 4356; 4. sour milk appearance 25# (adopt traditional zymotic breast) from Tibet; 5. sour milk appearance 36# (adopt spontaneous fermentation breast) from Tibet; 6.L.casei ATCC393; 7.L.rhamnosus1.2134; 8.L.plantarum 1.2437; 9.L.fermentum 1.1880.

Fig. 2, real time fluorescent quantitative detect the typical curve of Lactobacterium acidophilum.

Fig. 3, real time fluorescent quantitative detect the unimodal figure of melt curve analysis of Lactobacterium acidophilum.

[embodiment]

Below in conjunction with embodiment the present invention is described further.

Embodiment 1

The fast qualitative of Lactobacterium acidophilum detects in the probiotic bacteria milk product:

This embodiment use contains purpose and increases pulsating plasmid pAT as positive control; DH ₂O makes negative control; The traditional zymotic yoghourt appearance 25# that use L.acidophilus ATCC 4356 in addition, adopts from Tibet, spontaneous fermentation yoghourt appearance 36#, L.casei ATCC393, L.rhamnosus 1.2134 and the L.plantarum 1.2437 that adopts from Tibet carry out as sample.

Described L.rhamnosus 1.2134, L.plantarum 1.2437 are available from Chinese common micro-organisms DSMZ; L.acidophilus ATCC 4356, L.casei ATCC393 are available from U.S. American Type Culture Collection.

A. the preparation of template DNA

Adopt frozen-thawed-CTAB method extracting sample DNA, concrete steps are following:

The milk-acid bacteria (comprising pattern bacterium L.acidophilus ATCC 4356) of (1) getting 0.5g probiotics fermention breast sample or MRS culture medium culturing is 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 L5M 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.

(3) 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;

(4) draw the upper strata water, add with the isopyknic above-mentioned benzene chloroform of said water and iso pentane alcohol mixture extracting once, separation of supernatant;

(5) in described supernatant, 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 deposition with the centrifugal 5min of 10000g/min;

(6) said deposition is with 70 volume % aqueous ethanolic solutions washing 2 times, and seasoning at room temperature obtains template DNA again; Add the aseptic ultrapure water Hui Rong of 50.0 μ L then, be equipped with temperature-20 ℃ following a 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 (Af and Ar), 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 L.acidophilus ATCC 4356 is connected conversion DH5 α with pMD18, obtains the sub-pAT of positive colony.

D. result and judgement

Through the amplification of the species specificity primer PCR of L.acidophilus, the agarose gel electrophoresis assay is seen Fig. 1, and swimming lane 1 increases pulsating plasmid pAT as positive control to contain purpose, swimming lane 2 with aqua sterilisa as negative control.It is generally acknowledged that the expection amplified band appears in positive characteristic at the 759bp place, explain in this sample and contain L.acidophilus that negative control does not have the characteristic band.Swimming lane 3 is pattern bacterium L.acidophilus ATCC 4356; Spontaneous fermentation breast 25#, the 36# sample of swimming lane 4,5 for adopting from Tibet, the result is negative, explains that this 2 increment does not originally contain L.acidophilus.Swimming lane 6～9 is outer other kinds milk-acid bacteria of L.acidophilus, and the result is negative, explains that primer specificity is good, and is consistent with expected results.

Embodiment 2.

The quantitatively determined of Lactobacterium acidophilum in the probiotic bacteria milk product:

Quantitatively determined to business-like Lactobacterium acidophilum NCFM.Its determination step is following:

A. the preparation of the total RNA of sample

Adopt the Trizol method to extract: after cultured milk prod 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 through 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 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

Quantitative fluorescent PCR is according to SYBR PrimeScript ^TMRT-PCR Kit (the precious biotechnology in Dalian ltd, 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 L10 μ mol/L upstream and downstream primer (Af and Ar), 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, 56 ℃ 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 pAT 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 Lactobacterium acidophilum with the initial cycle number (Ct) that arrives fluorescence threshold in the PCR reaction process, sees accompanying drawing 2.

F, result and analysis

Visible by Fig. 2, Ct value and copy number are better linearity and concern, are X-coordinate with the logarithm of original template amount; The Ct value is an ordinate zou; The quantitative fluorescent PCR typical curve equation of drawing out: Y=-3.582X+38 (Y represents the Ct value, and x represents the logarithm of original template amount), linearly dependent coefficient is 0.987 between original template concentration and the Ct value; Slope is-3.582, and the L.acidophilus typical curve of being set up meets the quantitative requirement of Real-Time PCR.Visible by Fig. 3, the melt curve analysis of amplified production is consistent, and is the curve of simple spike type all, proves that the quantitative reaction systemic characteristic of optical dye is good, and 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 the Lactobacterium acidophilum of describing with embodiment 1 carries out the fluorescent quantitative PCR of gene fragment of the 16S rRNA of Lactobacterium acidophilum simultaneously with identical system.Through typical curve and the iQ that adopts Bio-Red company ^TMThe fast quantification that the computer software analysis of 5 multiple real time fluorescence quantifying PCR appearance carries out Lactobacterium acidophilum in the probiotic bacteria milk product detects.The mensuration result of these samples and analytical error result are listed in the table below in 1 in the lump.

The Real-time of table 1 L.acidophilus fermented-milk sample measures the result

The fermentation of sample list bacterium	X+SD copy number/g fermented-milk
		L.acidophilus?NCFM	7.9±0.8

Result by table 1 clearly illustrates that, quantitative PCR can be accurately quantitative living lactobacillus acidophilus in the fermented-milk.

Sequence table

< 110>Agricultural University of the Inner Mongol

< 120>fast qualitative of Lactobacterium acidophilum, method for quantitatively determining in the probiotic bacteria milk product

<130>

<160>2

<210>1

<211>20

<212>DNA

< 213>artificial sequence

<220>

< 223>to the description of artificial sequence: the upstream primer Af of institute of the present invention design and use.

<400>1

AGCTGAACCA ACAGATTCAC 20

<210>2

<211>20

<212>DNA

< 213>artificial sequence

<220>

< 223>to the description of artificial sequence: the downstream primer Ar of institute of the present invention design and use.

<400>2

ACTACCAGGG?TATCTAATCC 20

Claims (3)

1. the fast qualitative measuring method of Lactobacterium acidophilum in the probiotic bacteria milk product is characterized in that the step of this method is following:

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, in shaking table, under 37 ℃ of temperature, shake 4h with 200r/min in mass volume ratio 10%SDS and 10.0 μ L 10mg/mL Proteinase Ks;

(3) add 100.0 μ L 5M NaCl and 100.0 μ L CTAB/NaCl solution, 65 ℃ of water-bath 10min; Said CTAB/NaCl solution is that 4.1g NaCl is dissolved in the 80ml water, slowly adds the solution that 10g CTAB obtains again;

(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 said water, chloroform and iso pentane alcohol mixture extracting once, separation of supernatant;

(6) in described supernatant, 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 deposition with the centrifugal 5min of 10000g/min;

(7) said deposition use volume ratio is 70% aqueous ethanolic solution washing 2 times, and seasoning at room temperature obtains template DNA again; Add the aseptic ultrapure water Hui Rong of 50.0 μ L then, subsequent use 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 L dna profilings, 0.25uL 5U/ μ L Taq enzyme, redistilled water complements to 25.0 μ L;

The upstream and downstream primer is respectively:

Upstream primer Af is 5 '-AGCTGAACCAACAGATTCAC-3 ',

Downstream primer Ar is 5 '-ACTACCAGGGTATCTAATCC-3 ', and the amplification fragment length is 759bp.

PCR reaction conditions: 95 ℃ of preparatory sex change 3min; 95 ℃ of sex change 40S, 57 ℃ 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 DNA as the positive contrast of template, with aqua sterilisa as the negative contrast of template; According to expection characteristic band occurs at the 759bp place, confirm to contain Lactobacterium acidophilum in this probiotic bacteria milk product, then do not contain Lactobacterium acidophilum on the contrary.

2. method according to claim 1, it is characterized in that containing the purpose pulsating DNA that increases is to be connected positive colony that transformed into escherichia coli DH5 α obtains with pMD18 through the PCR product with L.acidophilus ATCC4356.

3. the quantitative determination method of Lactobacterium acidophilum in the probiotic bacteria milk product is characterized in that the step of this method is following:

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 in filling the mortar of liquid nitrogen, grinding, and 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 appearance to measure concentration; Adopt 1% agarose gel electrophoresis to detect the RNA integrity, then sample is stored in-80 ℃;

B, post transcription cloning

5 * PrimerScript Buffer of reaction system 10.0 μ L:2.0 μ L TakaRa DRR063A, 0.5 μ L PrimerScript RT Enzyme Mix I, 0.5 μ L, 100 μ M Random 6mers, less than the Total RNA of 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

Premix Ex Taq ^TM, each 0.5 μ L10 μ mol/L upstream and downstream primer Af and Ar, 2.0 μ L cDNA templates, 9.5 μ L dH ₂O;

Quantitative fluorescent PCR reaction parameter: 95 ℃ of sex change 25S; 95 ℃ of sex change 10S, 56 ℃ 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 pAT of Lactobacterium acidophilum type strain ATCC4356 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, and counting Ct with the initial cycle that arrives fluorescence threshold in the PCR reaction process is the typical curve that ordinate zou obtains Lactobacterium acidophilum, as the reference standard of testing sample quantitatively determined;

E, result and judgement

DNA with testing sample cDNA and standard substance is a template, with the species-specific primer of Lactobacterium acidophilum, carries out the fluorescent quantitative PCR of gene fragment of the 16S rRNA of Lactobacterium acidophilum simultaneously with identical system; Obtain the starting template amount of Lactobacterium acidophilum in the probiotic bacteria milk product through the Ct value of typical curve and testing sample;

Said Lactobacterium acidophilum species specificity primer sequence is following:

Upstream primer Af is 5 '-AGCTGAACCAACAGATTCAC-3 ',

Downstream primer Ar is 5 '-ACTACCAGGGTATCTAATCC-3 ', and the amplification fragment length is 759bp.

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