CN105349669B - Fast qualitative, immue quantitative detection reagent box and the detection method of Bifidobacterium for being added in feed and application - Google Patents
Fast qualitative, immue quantitative detection reagent box and the detection method of Bifidobacterium for being added in feed and application Download PDFInfo
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Abstract
The invention discloses the fast qualitative of the Bifidobacterium for adding in feed, immue quantitative detection reagent box and detection method and applications, by pair it has been reported that Bifidobacterium genome sequence comparison analysis, the present invention provides primer 5 '-GTCATCTGCCCGACATCTACAA-3 ', the 5 '-TAGGCTTCAGAGCAACGCAAC-3 ' for Bifidobacterium qualitative and quantitative detection, the present invention provides a kind of preprocess methods of Feed Sample simultaneously, probiotics in feed is sufficiently discharged, the probiotics number in feed is accurately really reflected.The present invention can be on the basis of without probiotics pure culture, and easy, quickly and accurately Bifidobacterium in qualitative and quantitative analysis pannage, detection program is simple, detection efficiency is high, accuracy is good, and high sensitivity is reproducible.
Description
Technical field
The invention belongs to animal and veterinary technical fields.The bifid bar that more particularly, the present invention relate to add in feed
Fast qualitative, immue quantitative detection reagent box and the detection method of bacterium and application.
Background technique
With the disabling of antibiotic in China's animal feed, novel green additive is developed into hot spot.Benefit at present
Probiotics preparation continues to develop, but in the method for its qualitative and quantitative analysis science not enough and quickly, this is limited to a certain extent
The development of probiotics preparation is made.Generally use the tradition side of biochemical reactions and the counting of selective medium isolation of pure culture
Method, easily because detection efficiency caused by strain difference is limited, testing result is unstable, in addition to this, conventional method can also consume
Take a large amount of time and resource.Real-time fluorescence quantitative PCR is the leap of qualitative to quantitative, Yin Qite for round pcr
Anisotropic strong, reproducible, accurate and efficient feature becomes molecular biology and the very important quantitative inspection of microbiological art
Survey method.By species specificity PCR and Real-Time Fluorescent Quantitative PCR Technique, establish the fast qualitative of Bifidobacterium in pannage,
Quantitative detecting method can be improved detection efficiency, simplify detection program, and promotes the development of feedstuff industry and aquaculture, for benefit
The quick and long term growth of raw bacterium and feed industry provides technical guarantee.
The application for the Bifidobacterium contained in pannage, by pair it has been reported that Bifidobacterium genome sequence
Analysis is compared, the conservative gene of Bifidobacterium gene as a purpose, the kind of the gene order of designed, designed Bifidobacterium are chosen
Specific primer carries out species specificity PCR and real-time fluorescence quantitative PCR using the primer, passes through agarose gel electrophoresis figure
Spectrum analysis and real-time fluorescence quantitative PCR atlas analysis establish the fast qualitative of Bifidobacterium and quantitative determination side in pannage
Method.
Summary of the invention
Fast qualitative, the immue quantitative detection reagent box of Bifidobacterium for being added in feed, including primer: 5 '-
GTCATCTGCCCGACATCTACAA-3’、5’-TAGGCTTCAGAGCAACGCAAC-3’。
It is another object of the present invention to provide the fast qualitatives of the Bifidobacterium for adding in feed, quantitative inspection
The detection method of test agent box, method is simple, and repeatability is high, as a result accurately.
It is another object of the present invention to provide a kind of fast qualitative of Bifidobacterium for being added in feed, determine
Measure the application of detection kit.
In order to achieve the above object, the present invention takes following technical measures:
Fast qualitative, the immue quantitative detection reagent box of Bifidobacterium for being added in feed, including primer: 5 '-
GTCATCTGCCCGACATCTACAA-3’、5’-TAGGCTTCAGAGCAACGCAAC-3’。
The detection method of the fast qualitative of Bifidobacterium for being added in feed, immue quantitative detection reagent box, including feed
The method of sample pretreatment, this method comprises:
Feed 25g by sufficiently crushing mixing is mixed with the sterile saline of 225mL, at 4 DEG C with 100 revs/min
Speed shakes 1-2h, is made into 10% uniform dilution.10 times are carried out to uniform dilution and is incremented by dilution, selects 2 or more to fit
Suitable dilution extracts bacterial genomes DNA or RNA as needed.
The application of the fast qualitative, immue quantitative detection reagent box of Bifidobacterium for adding in feed, application include benefit
It is prepared into the detection reagent for Bifidobacterium with the kit, or carries out the qualitative detection of Bifidobacterium using the kit,
Or the quantitative detection of Bifidobacterium is carried out using the kit, or carry out the qualitative of Bifidobacterium simultaneously using the kit and determine
The detection of amount.
Compared with prior art, the invention has the following advantages that
(1) processing method of feed sample of the present invention makes in the extraction process of probiotics, probiotics
Expand limited, while the probiotics in feed can sufficiently discharge again, and value measured in this way could be reflected really in feed
Probiotics number, be a species specific Bifidobacterium extracting method suitable for feed, for the present invention initiate.
(2) present invention at present it has been reported that Bifidobacterium and feed in may addition such as lactobacillus acidophilus, plant
All genome sequences of other probiotics such as lactobacillus, lactobacillus bulgaricus, Lactobacillus casei, enterococcus faecium, enterococcus faecalis
Column have carried out comparing analysis, have selected conservative single copy gene gene as a purpose, and with the design of the conserved sequence of the gene
The primer of specificity carries out qualitative and quantitative analysis with the Bifidobacterium in the primer pair feed, and acquired data could be true
The quantity for reflecting the Bifidobacterium added in feed.
(3) present invention can be on the basis of without probiotics pure culture, easy, quickly and accurately qualitative, quantitative inspection
Bifidobacterium in pannage is surveyed, whole process is simple to the detection program of Bifidobacterium in feed, detection efficiency is high, accuracy
Good, high sensitivity, lowest detection standard is more much lower than traditional method, and reproducible.
Detailed description of the invention
Fig. 1 is the qualitative detection schematic diagram of Bifidobacterium.
M:DL2000marker；1: Lactobacillus casei；2: lactobacillus acidophilus；3: lactobacillus bulgaricus；4: enterococcus faecium；
5: enterococcus faecalis；6: bacillus coagulans；7: the feed added with animal bifidobacteria；8: positive control (contains target gene
Plasmid be template)；9: animal bifidobacteria；10: Escherichia coli；11: Lactococcus lactis；12 negative controls
Fig. 2 is verifying schematic diagram of the Real-time PCR to Bifidobacterium primer specificity.
Wherein A. acidophilus strain B lactobacillus plantarum C. lactobacillus bulgaricus D. Lactobacillus casei E. enterococcus faecalis F.
Enterococcus faecium G. Bifidobacterium H. Escherichia coli
Fig. 3 is various concentration Bifidobacterium standard strain amplification curve schematic diagram.
Wherein A.10-1Dilution；B.10-2Dilution；C.10-3Dilution；D.10-4Dilution；E.10-5Dilution；F.10-6Dilution；
G.10-7Dilution；H.10-8Dilution.
Fig. 4 is Bifidobacterium quantitative measurement standard curve synoptic diagram.
Specific embodiment
Technical solution described in the embodiment of the present invention is if not otherwise specified the ordinary skill in the art.Agents useful for same or
Material discloses.
PMD18-T cloning vector core DH5 α competent cell is purchased from Takara Biotechnology Co., Ltd, Trizol examination
Agent is the product that the RNA of extraction is specialized in by Invitrogen company；DNase I is TaKaRa Products；It is used during this
The reagents such as chloroform, isopropanol, dehydrated alcohol be the production of three factory of Tianjin chemical reagent product.The purchase of Bifidobacterium standard strain
From China General Microbiological culture presevation administrative center.
Embodiment 1:
The selection of Bifidobacterium purpose detection gene:
The present invention at present it has been reported that Bifidobacterium and feed in may addition such as lactobacillus acidophilus, Bulgaria
Other probiotics such as lactobacillus, lactobacillus plantarum, lactobacillus bulgaricus, Lactobacillus casei, enterococcus faecium, enterococcus faecalis are all
Genome sequence carried out a large amount of comparisons analysis, select the target gene of qualitative and quantitative analysis, the target gene is in bifid bar
It is conservative house-keeping gene in bacterium genome, and is single copy gene, specificity is devised with the conserved sequence of the gene
Primer carries out qualitative and quantitative analysis to the Bifidobacterium in feed, and acquired data could be reflected really in feed and be added
Bifidobacterium quantity.For the primer of target gene design are as follows: 5 '-GTCATCTGCCCGACATCTACAA-3 ', 5 '-
TAGGCTTCAGAGCAACGCAAC-3, the primer can be used for the qualitative and quantitative detection of Bifidobacterium.
Embodiment 2:
Fast qualitative, the immue quantitative detection reagent box of Bifidobacterium for being added in feed, including primer: 5 '-
GTCATCTGCCCGACATCTACAA-3’、5’-TAGGCTTCAGAGCAACGCAAC-3。
Embodiment: 3:
The detection method of the fast qualitative of Bifidobacterium for being added in feed, immue quantitative detection reagent box, including feed
The method of sample pretreatment, this method comprises:
Feed 25g by sufficiently crushing mixing is mixed with the sterile saline of 225mL, at 4 DEG C with 100 revs/min
Speed shakes 1-2h, is made into 10% uniform dilution.Sterile working carries out 10 times to uniform dilution prepared by previous step and passs
Increase dilution, selects 2~3 or more suitable dilutions, as needed, extract bacterial genomes DNA or RNA.
Remaining step carries out qualitative and quantitative identification using DNA or RNA of the primer pair in embodiment 1 to extraction.
The detection method of the fast qualitative of enterococcus faecium for adding in feed, immue quantitative detection reagent box, specifically includes
Following steps:
A, the preparation of template DNA
The sterilizing that sterile working will be put into the sterile saline containing 225mL by sufficiently crushing the feed 25g mixed
In wide-mouth bottle, (4 DEG C) shake 1.5h with 100 revs/min of speed under cryogenic, are made into 10% uniform dilution.It is sterile
It operates and 10 times of incremental dilutions is carried out to uniform dilution prepared by previous step, 3 or more suitable dilutions are selected, using liquid nitrogen
Freeze thawing-CTAB method extracts sample DNA:
(1) it takes pretreated Feed Sample in 2.0mL centrifuge tube, 500 μ L TE buffers is added after mixing,
It is placed in liquid nitrogen and freezes, taken out after freezing and be placed on water-bath 5min in 65 DEG C, multigelation 4 times；
(2) after freeze thawing, it is charged with the SDS and 10.0 μ L110mg/mL Proteinase Ks of 60.0 μ L10%, 37 DEG C are shaken
Bed vibrates 4h with 200r/min；
(3) 100.0 μ L5MNaCl and 100.0 μ L CTAB/NaCl, water-bath 10min in 65 DEG C is added；
(4) then, the solution obtained with step (3) isometric phenol, chloroform and iso pentane alcohol mixture is added, they
Volume ratio is respectively 25:24:1, after mixing with 10000g/min be centrifuged 10min, the solution be divided into upper strata aqueous phase, middle layer solid phase and
Lower layer's organic phase；
(5) upper strata aqueous phase is drawn, the phenol equal with water phase volume, chloroform and iso pentane alcohol mixture extracting is added once,
Separate supernatant；
(6) be added into above-mentioned supernatant 1% volume 3mol/L sodium acetate and 1 times of volume and isopropanol, after mixing
It is stored at room temperature 30min, 10000g/min is centrifuged 5min, discards supernatant；
(7) twice with 70% ethanol washing precipitating, template DNA is obtained after spontaneously drying at room temperature, it is sterile that 50.0 μ L are added
Ultrapure water dissolving DNA, -20 DEG C save backup.
As the template of next step qualitative PCR detection, saved backup at -20 DEG C.
B, PCR amplification
25.0 μ L of reaction system:
2.5 μ 10 × PCR of L Buffer (contain Mg2+)、
2.0 μ L 10mmol/L DNTPs (each 2.5mmol),
1.0 μ L 10mmol/L upstream and downstream primers,
1.0 μ L 50ng/L DNA profilings,
0.5 μ L 5U/L Taq enzyme,
Use ddH2O complements to 25.0 μ L；
PCR reaction condition: 95 DEG C of initial denaturation 3min；95 DEG C of initial denaturations 30s, 64 DEG C of annealing 30s, 72 DEG C of extension 45s, circulation
40 times；72 DEG C of extension 10min, obtain pcr amplification product, save at 4 DEG C；
5.0 μ L PCR products are taken to be detected using 1.0% agarose gel electrophoresis.Agarose gel electrophoretogram point
Analysis is to be carried out using Shanghai day with the instrument that trade name Tanon-4100 gel electrophoresis imaging system is sold；
The Bifidobacterium upstream and downstream specific primer is respectively:
Upstream primer are as follows: 5 '-GTCATCTGCCCGACATCTACAA-3 '
Downstream primer are as follows: 5 '-TAGGCTTCAGAGCAACGCAAC-3 '
C, result and judgement
Set when detection using the Plasmid DNA containing purposive amplified fragments as template as positive control, using aqua sterilisa as
Template is negative control；It is expanded simultaneously using the non-control strain of the same race of each reference culture as reference.If reference culture
There is the specific band being consistent with expected size in amplified production, and the spy do not occur in negative control and non-control strain of the same race
Anisotropic band then shows that primer is special.According to occurring expected characteristic bands at 256bp, show in the feed containing double
Discrimination bacillus does not contain Bifidobacterium then on the contrary.
The Plasmid DNA of the amplified fragments containing target gene be by by the PCR product of Bifidobacterium with
The positive clone molecule that pMD18T carrier link conversion escherichia coli DH5a obtains.
The qualitative detection is the electrophoresis carried out under the voltage of 5V/cm, and the time is about 20-30min, then is used
Goldvie w dyeing, then carries out uv photography.
D, the extraction of RNA
(1) preparation of sample total serum IgE
Extract the total serum IgE in Feed Sample using Trizol method: sterile working will be by sufficiently crushing the feed 25g mixed
It is put into the sterilizing wide-mouth bottle of the sterile saline containing 225mL, (4 DEG C) are shaken under cryogenic with 100 revs/min of speed
1.5 hours are swung, 10% uniform dilution is made into.Sterile working carries out 10 times to uniform dilution prepared by previous step and is incremented by
Dilution, the dilution after selecting 3 or more suitable dilutions add rapidly in feed dilution for extracting total serum IgE
Enter 1mLT rizol reagent, by Trizol kit (D9108A) specification extract sample RNA, RNA extract the step of it is as follows:
1. chloroform (1/5 volume of RNAiso Plus, 200L) is added into the homogenate lysate of above-mentioned steps, cover tightly
Centrifuge tube lid is shaken vigorously by hand for 15 seconds when oscillation (chloroform low boiling point, volatile, careful centrifuge tube lid is answered to flick suddenly).To
After solution fully emulsified (no phase separation phenomenon), then it is stored at room temperature 5min.
2. 4 DEG C of centrifugation 15min of 12,000g.
3. carefully taking out centrifuge tube from centrifuge, homogenate is divided into three layers at this time, it may be assumed that colourless supernatant, intermediate
White egg white and with coloured lower layer's organic phase.Aspirate supernatant (usually taking 450L) be transferred to another new 1.5mL from
(white middle layer is never sucked out) in heart pipe
4. isometric isopropanol (450L) is added into supernatant, after the centrifuge tube that turns upside down mixes well, 15~30
10min is stood in DEG C environment.
5. 4 DEG C of centrifugation 10min of 12,000g.Generally after centrifugation, test tube bottom will appear precipitating.
It carefully discards supernatant, 75% ethyl alcohol 1mL (being sure not to touch precipitating) is slowly added along centrifugation tube wall, gently up and down
Washing centrifuge tube tube wall is overturned, carefully discards ethyl alcohol (in order to preferably control the salt in RNA after 12,000g 4 DEG C of centrifugation 5min
Ion concentration, should ethyl alcohol cleared as far as possible).Reusable 75% ethyl alcohol 1mL is cleaned one time.
Drying at room temperature precipitates 5~10min (cannot be centrifuged or heat drying, otherwise RNA will be difficult to dissolve), is added suitable
The RNase-free water (enteron aisle usually takes 50L, and cell, bacterium take 20L) of amount dissolves precipitating, can gently be blown with liquid-transfering gun when necessary
Precipitating is beaten, is completely dissolved and is placed on ice to RNA precipitate.
(2) Concentration Testing of RNA, dilution
1. the foundation of background: taking RNase-free water 2L, established on trace dna Protein Detection instrument NanoDrop 2000
Blank；The RNA concentration for detecting RNase-free water, is advisable in ± 5ng/L.
2. test sample concentration: tissue sample concentration twice surveyed twice with being advisable no more than 50ng/L by detectable concentration difference
The average value of amount is sample RNA concentration.
③1.7<A260/A280<2.1；Genetic chip additional conditions: A260/A230 > 2.0
4. the dilution of RNA sample:
Take X L sample to dilute, then without enzyme water additive amount: without enzyme water volume Y=(original liquid concentration/600-1) * X (L), cell,
Bacteria samples do not need generally to dilute.
5. checking again for RNA concentration after dilution, it is advisable with 600 ± 50ng/L.
Ensure to completely eliminate the DNA pollution in RNA, the RNA sample purified is surveyed using micro UV detector
Determine concentration, using the integrality of 1.0% agarose gel electrophoresis detection RNA, is then stored in sample spare at -80 DEG C；
E, post transcription cloning
10 μ L:2.0 μ 5 × PrimeScript of L Buffer of reaction system 2 (for Real Time), 0.5 μ L
PrimeScript RT Enzyme Mix I、0.5μL RT Primer Mix、0.5uL Random Primers(100uM)、
Total RNA (< 500ng), RNase Free dH2O complements to 10.0 μ L.
Post transcription cloning reaction condition: the reaction solution of mixing is placed in PCR instrument after 37 DEG C of heat preservation 15min, and 85 DEG C add
Hot 5s finally by reverse transcription at cDNA be cooled to 4 DEG C of preservations.The cDNA of synthesis is stored in -80 DEG C of refrigerators.
F, real-time fluorescence quantitative PCR
25.0 μ L:12.5 μ L of PCR reaction systemEx TaqTM, the 10 μm of upstream and downstream ol/L primers are each
0.5 μ L, cDNA template 2.0 μ L, dH2O9.5μL。
Quantitative fluorescent PCR response parameter: 95 DEG C of initial denaturation 3min；95 DEG C of denaturation 30s, 64 DEG C of annealing 30s, 72 DEG C extend
45s is recycled 40 times, and each sample is repeated 3 times, and is saved under 4 DEG C of low temperature.
The Bifidobacterium upstream and downstream specific primer is respectively:
Upstream primer are as follows: 5 '-GTCATCTGCCCGACATCTACAA-3 '
Downstream primer are as follows: 5 '-TAGGCTTCAGAGCAACGCAAC-3 '
The preparation of outer standard items and the drafting of standard curve
The preparation of outer standard items: it extracts containing with the plasmid of the amplified fragments of the target gene of Bifidobacterium type strain
DNA after ultraviolet specrophotometer measures A value, calculates copy number, carries out 10 times and be serially diluted, gradient dilution to 109-102It copies
Shellfish number/uL concentration carries out quantitative fluorescent PCR reaction in this, as outer standard items；
The drafting of standard curve: using the logarithm of the positive template of different copy numbers as abscissa, to be reached in PCR reaction process
Initial cycle number (Ct) to threshold value is that ordinate obtains the standard curve of Bifidobacterium, the ginseng as sample to be tested quantitative determination
Sighting target is quasi-.
G, result and judgement
Using the DNA of sample to be tested cDNA and standard items as template, with the species-specific primer of Bifidobacterium, with identical body
System at the same carry out Bifidobacterium target gene fragment fluorescent quantitative PCR, pass through the Ct of standard curve and sample to be tested
Value finds out the starting template amount of Bifidobacterium in Feed Sample.
Embodiment 4:
Kit specific detection:
1) specificity verification of Bifidobacterium qualitative detection:
The primer and method provided using embodiment 3 to lactobacillus acidophilus, lactobacillus plantarum, lactobacillus bulgaricus, is done
Lactobacillus paracasei, enterococcus faecium, enterococcus faecalis, Escherichia coli, positive control (plasmid containing target gene is template), animal are double
Discrimination bacillus, the feed added with animal bifidobacteria；Negative control (aqua sterilisa) carries out PCR amplification.
As the result is shown: only swimming lane 7 (feed added with animal bifidobacteria), the 8 positive controls (matter containing target gene
Grain is template), 9 (animal bifidobacterias) produce the amplified band of the 256bp of specificity, and other bacterial strains do not have, show
Primer provided by the invention only can specificity detection Bifidobacterium.There is the amplification of swimming lane 7 it is found that of the invention mentions simultaneously
The sample pretreating method of confession can carry out separation and Extraction (Fig. 1) to Bifidobacterium well.
2) Bifidobacterium quantitative detection specificity verification
The method provided using embodiment 3 carries out Bifidobacterium quantitative detection special with non-bacterium of the same race as reference strain
Opposite sex verifying.
Reference strain used has: lactobacillus acidophilus；Lactobacillus plantarum；Lactobacillus bulgaricus；Lactobacillus casei；Dung intestines
Coccus；Enterococcus faecalis；Bifidobacterium adolescentis.
As a result judge: 1. cycle threshold (Ct value)≤35 shows that PCR has the amplification of target dna in the process, can directly determine
For the positive；2. real-time fluorescent PCR amplification should be reformed between 32~40 to sample genetic test Ct value；It is outer after expanding again
Source gene C t value is still less than 40, and curve has obvious increased logarithmic phase, and negative control, positive control and blank control knot
Fruit is normal, then can determine that as the positive；Foreign gene Ct value is greater than 40, and negative control, positive control and blank after expanding again
Results of comparison is normal, can determine that as feminine gender.
The result shows that: template be different strains DNA, primer be Bifidobacterium specific primer under conditions of, only in mould
Plate be doubly-odd nucleus under the conditions of, can just obtain the amplification curve with obvious increased logarithmic phase, i.e. Bifidobacterium quantitative detection
With specific (Fig. 2)
Embodiment 5:
The detection of kit sensitivity:
Bifidobacterium is cultivated into certain time in the medium, its A value is surveyed, estimates its bacterium number, then presses 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7, 10-8Multiple dilution, take the bacterium solution of last several gradients to apply plate count, be repeated 3 times, take it
Average value determines its true strain density.After each gradient bacterium solution respectively takes 3 pipes extraction RNA to be reversed to cDNA simultaneously, to test sample
Product cDNA is template, and the purpose base of Bifidobacterium is carried out with the species-specific primer of Bifidobacterium described in embodiment 3 and method
Because of the fluorescent quantitative PCR (Fig. 3) of segment, and using the logarithm of plate count total bacteria count as abscissa, quantitative fluorescent PCR is anti-
The Ct value answered is ordinate, obtains the standard curve (Fig. 4) of bacterium number Yu Ct value.
The result shows that: corresponding total bacteria count is the Monitoring lower-cut of Bifidobacterium when Ct value is 32, as 4.7 ×
104Cfu, that is, the lowest detection standard of the fluorescent PCR detection of primer provided by the invention.
Embodiment 6:
In the feed without Bifidobacterium, addition final concentration of 4.07 ± 0.15 × 108The Bifidobacterium of cfu/g feed
(plate count bacterium number is 3.9 × 108Cfu/g feed, 4.2 × 108Cfu/g feed, 4.1 × 108Cfu/g feed), utilize this hair
Bright method is detected, obtained real-time quantitative PCR detection Ct value be 18.68 ± 0.30 (Ct value is respectively 19.01,
18.44,18.59) the standard curve y=-3.4921x+48.612 (R, established using Bifidobacterium standard items2=0.9947),
Obtain be in feed Bifidobacterium concentration be 3.77 ± 0.70 × 108Cfu/g feed (theoretical bacterium number is respectively 3.0 ×
108Cfu/g feed, 4.4 × 108Cfu/g feed, 4.0 × 108Cfu/g feed), it is statisticallyd analyze through SPSS 17.0, plate count
Two groups of data differences are obtained not significantly (P=0.565) with method of the invention, i.e., sample pretreating method combination institute of the invention
Target gene is chosen, testing result has accuracy.
SEQUENCE LISTING
<110>Wuhan Polytechnic University
<120>fast qualitative, immue quantitative detection reagent box and the detection method of the Bifidobacterium for adding in feed and
Using
<130>fast qualitative, immue quantitative detection reagent box and the detection method of the Bifidobacterium for adding in feed and
Using
<160> 2
<170> PatentIn version 3.1
<210> 1
<211> 22
<212> DNA
<213>artificial sequence
<400> 1
gtcatctgcc cgacatctac aa 22
<210> 2
<211> 21
<212> DNA
<213>artificial sequence
<400> 2
taggcttcag agcaacgcaa c 21
Claims (1)
1. application of the kit in the qualitative and quantitative detection of Bifidobacterium, the kit includes primer 5 '-
GTCATCTGCCCGACATCTACAA-3',5'-TAGGCTTCAGAGCAACGCAAC-3'.The application method of the kit, packet
The preprocess method of Feed Sample is included, this method comprises: by the sterile physiological for sufficiently crushing the feed 25g and 225mL that mix
Salt water mixing shakes 1-2h at 4 DEG C with 100 revs/min of speed, is made into 10% uniform dilution；Uniform dilution is carried out
10 times are incremented by dilution, select 2 or more dilutions, as needed, extract bacterial genomes DNA or RNA.
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| CN101319250A (en) * | 2007-06-06 | 2008-12-10 | 东北农业大学 | Method for quick quantitative determination of active bifidobacteria |
| CN101712988A (en) * | 2009-09-21 | 2010-05-26 | 内蒙古农业大学 | Method for quickly, qualitatively and quantitatively measuring Bifidobacteria in probiotic dairy products |
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