CN105349670B - For fast qualitative, immue quantitative detection reagent box and the detection method of Lactobacillus casei added in feed and application - Google Patents
For fast qualitative, immue quantitative detection reagent box and the detection method of Lactobacillus casei added in feed and application Download PDFInfo
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Abstract
The invention discloses fast qualitative, immue quantitative detection reagent box and the detection method of Lactobacillus casei for being added in feed and applications, by pair it has been reported that Lactobacillus casei genome sequence comparison analyze, the present invention provides 5 ' TGCGGCGGTAAAGGTTG of primer, 3 ', 5 ' CGTCTGTGTAGAAACTGCGAATG 3 ' for Lactobacillus casei qualitative and quantitative detection, the present invention provides a kind of preprocess methods of Feed Sample simultaneously, probiotics in feed is fully discharged, accurately really reflects the probiotics number in feed.The present invention can be on the basis of without probiotics pure culture, and easy, quickly and accurately Lactobacillus casei 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 cheese breast that more particularly, the present invention relate to be added in feed
Fast qualitative, immue quantitative detection reagent box and the detection method of bacillus and application.
Background technology
With the disabling of antibiotic in China's animal feed, new green additive is developed into hot spot.Benefit at present
Probiotics preparation continues to develop, but in the method science not enough of its qualitative and quantitative analysis and quick, this is limited to a certain extent
The development of probiotics preparation is made.The tradition side that generally use biochemical reactions and selective medium isolation of pure culture count
Method, easily because detection efficiency caused by strain difference be limited, testing result it is unstable, in addition, conventional method can also consume
Take substantial amounts of time and resource.Real-time fluorescence quantitative PCR is the leap of qualitative to quantitative for round pcr, Yin Qite
The characteristics of opposite sex is strong, reproducible, accurate and efficient 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 quick of Lactobacillus casei in pannage and determine
Property, quantitative detecting method, detection efficiency can be improved, simplify detection program, and promote the development of feedstuff industry and aquaculture, be
The quick and long term growth of probiotics and feed industry provides technical guarantee.
The application is directed to the Lactobacillus casei that contains in pannage, by pair it has been reported that Lactobacillus casei genome sequence
The comparison analysis of row, chooses the conservative gene of Lactobacillus casei gene as a purpose, the gene sequence of designed, designed Lactobacillus casei
The species specificity primer of row carries out species specificity PCR and real-time fluorescence quantitative PCR using the primer, is coagulated by agarose
Gel electrophoresis atlas analysis and real-time fluorescence quantitative PCR atlas analysis, establish in pannage the fast qualitative of Lactobacillus casei and
Method for quantitatively determining.
The content of the invention
Fast qualitative, immue quantitative detection reagent box for the Lactobacillus casei added in feed, including primer：5’-
TGCGGCGGTAAAGGTTG-3’、5’-CGTCTGTGTAGAAACTGCGAATG-3’。
It is another object of the present invention to provide be used for the fast qualitative of the Lactobacillus casei added in feed, quantify
The detection method of detection kit, 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 for the Lactobacillus casei that adds in feed,
The application of immue quantitative detection reagent box.
In order to achieve the above object, the present invention takes following technical measures：
Fast qualitative, immue quantitative detection reagent box for the Lactobacillus casei added in feed, including primer：5’-
TGCGGCGGTAAAGGTTG-3’、5’-CGTCTGTGTAGAAACTGCGAATG-3’。
The detection method of fast qualitative, immue quantitative detection reagent box for the Lactobacillus casei that is added in feed, including raising
Expect the method for sample pretreatment, this method includes：
It is mixed by the feed 25g for fully crushing mixing 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 of incremental dilutions are carried out to uniform dilution, select 2 or more to fit
Suitable dilution factor, as needed, extraction bacterial genomes DNA or RNA.
Application for the fast qualitative, immue quantitative detection reagent box of the Lactobacillus casei that are added in feed, application include
It is prepared into using the kit for the detection reagent of Lactobacillus casei or carries out the qualitative of Lactobacillus casei using the kit
Detection carries out the quantitative detection of Lactobacillus casei using the kit or is carried out at the same time Lactobacillus casei using the kit
Qualitative and quantitative detection.
Compared with prior art, the present invention has the following advantages：
(1) processing method of feed sample of the present invention causes in the extraction process of probiotics, probiotics
Expand limited, while the probiotics in feed can fully discharge again, and so measured value could be reflected really in feed
Probiotics number, be a species specific Lactobacillus casei extracting method suitable for feed, for the present invention initiate.
(2) present invention at present it has been reported that Lactobacillus casei and feed in may addition such as lactobacillus acidophilus, plant
All genome sequences of other probiotics such as object lactobacillus, lactobacillus bulgaricus, enterococcus faecium, enterococcus faecalis, Bifidobacterium
Row have carried out comparing analysis, have selected conservative single copy gene gene as a purpose, and have been designed with the conserved sequence of the gene
The primer of specificity carries out qualitative and quantitative analysis with the Lactobacillus casei in the primer pair feed, and acquired data could be true
The real quantity for reflecting the Lactobacillus casei added in feed.
(3) present invention can be on the basis of without probiotics pure culture, easy, quickly and accurately qualitative, quantitative inspection
Lactobacillus casei in pannage is surveyed, whole process is simple to the detection program of Lactobacillus casei in feed, detection efficiency is high, accurate
Good, the high sensitivity of property, lowest detection standard is more much lower than traditional method, and reproducible.
Description of the drawings
Fig. 1 is the qualitative detection schematic diagram of Lactobacillus casei.
M：DL2000marker：1:Positive control (plasmid containing target gene is masterplate)；2:Lactobacillus casei；3:Add
Added with the feed of Lactobacillus casei；4：Lactobacillus acidophilus；5：Lactobacillus plantarum；6:Lactobacillus bulgaricus；7:Enterococcus faecium；8:
Enterococcus faecalis；9:Bifidobacterium bifidum；10:Animal bifidobacteria；11:Escherichia coli；12 negative controls
Fig. 2 is verification schematic diagrames of the Real-time PCR to Lactobacillus casei primer specificity.
Wherein A. acidophilus strain Bs lactobacillus plantarums C. lactobacillus bulgaricus D. Lactobacillus caseis E. excrement intestines balls
Bacterium F. enterococcus faecium G. Bifidobacterium H. bifidobacterium adolescentises.
Fig. 3 is various concentration Lactobacillus casei 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 Lactobacillus casei quantitative measurement standard curve synoptic diagram.
Specific embodiment
Technical solution described in the embodiment of the present invention is the ordinary skill in the art if not otherwise specified.Agents useful for same or
Material, it is disclosed.
PMD18-T cloning vector core DH5 α competent cells are purchased from Takara bio tech ltd, Trizol examinations
Agent is the product for the RNA that extraction is specialized in by Invitrogen companies；DNase I are TaKaRa Products；It is used during this
The reagents such as chloroform, isopropanol, absolute ethyl alcohol be the production of three factory of Tianjin chemical reagent product.Lactobacillus casei standard strain
Purchased from China General Microbiological culture presevation administrative center.
Embodiment 1：
Lactobacillus casei purpose detects the selection of gene：
The present invention at present it has been reported that Lactobacillus casei and feed in may addition such as lactobacillus acidophilus, Bao Jiali
Other probiotics such as sub- lactobacillus, lactobacillus plantarum, lactobacillus bulgaricus, enterococcus faecium, enterococcus faecalis, Bifidobacterium own
Genome sequence carried out a large amount of comparisons analysis, select the target gene of qualitative and quantitative analysis, the target gene is in cheese breast
It is conservative house-keeping gene in bacillus gene group, and is single copy gene, specificity is devised with the conserved sequence of the gene
Primer, qualitative and quantitative analysis is carried out to the Lactobacillus casei in feed, acquired data could be reflected really in feed
The quantity of the Lactobacillus casei of addition.For the target gene design primer be：5’-TGCGGCGGTAAAGGTTG-3’、5’-
CGTCTGTGTAGAAACTGCGAATG-3’；The primer can be used for the qualitative and quantitative detection of Lactobacillus casei.
Embodiment 2：
Fast qualitative, immue quantitative detection reagent box for the Lactobacillus casei added in feed, including primer：5’-
TGCGGCGGTAAAGGTTG-3’、5’-CGTCTGTGTAGAAACTGCGAATG-3’。
Embodiment:3：
The detection method of fast qualitative, immue quantitative detection reagent box for the Lactobacillus casei that is added in feed, including raising
Expect the method for sample pretreatment, this method includes：
It is mixed by the feed 25g for fully crushing mixing 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, select 2~3 or more suitable dilution factors, as needed, extraction bacterial genomes DNA or RNA.
Remaining step carries out qualitative and quantitative identification using the primer pair in embodiment 1 to the DNA or RNA of extraction.
The detection method of fast qualitative, immue quantitative detection reagent box for the Lactobacillus casei that is added in feed is specific to wrap
Include following steps：
A, the preparation of template DNA
Sterile working will be put into the sterilizing of the sterile saline containing 225mL by fully crushing the feed 25g of mixing
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 dilution factors are selected, using liquid nitrogen
Freeze thawing-CTAB methods extract sample DNA：
(1) pretreated Feed Sample is taken in 2.0mL centrifuge tubes, adds in 500 μ L TE buffer solutions 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, the SDS and 10.0 μ L110mg/mL Proteinase Ks of 60.0 μ L10% are charged with, 37 DEG C are shaken
Bed vibrates 4h with 200r/min；
(3) 100.0 μ L5MNaCl and 100.0 μ L CTAB/NaCl are added in, water-bath 10min in 65 DEG C；
(4) then, the solution obtained with step (3) isometric phenol, chloroform and iso pentane alcohol mixture are added in, they
Volume ratio is respectively 25:24:1,10min is centrifuged with 10000g/min after mixing, the solution be divided into upper strata aqueous phase, middle level solid phase and
Lower floor's organic phase；
(5) upper strata aqueous phase is drawn, adds in the phenol equal with water phase volume, chloroform and iso pentane alcohol mixture extracting once,
Separate supernatant；
(6) added in into above-mentioned supernatant 1% volume 3mol/L sodium acetates and 1 times of volume and isopropanol, after mixing
30min is stored at room temperature, 10000g/min centrifugation 5min discard supernatant；
(7) wash precipitation twice with 70% ethyl alcohol, template DNA is obtained after spontaneously drying at room temperature, it is sterile to add in 50.0 μ L
Ultra-pure 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：
Use ddH2O complements to 25.0 μ L；
PCR reaction conditions：95 DEG C of pre-degeneration 3min；95 DEG C of pre-degeneration 30s, 64 DEG C of annealing 30s, 72 DEG C extend 45s, cycle
40 times；72 DEG C of extension 10min, obtain pcr amplification product, are preserved at 4 DEG C；
5.0 μ L PCR products is 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 electrophoresises imaging system is sold；
The Lactobacillus casei upstream and downstream specific primer is respectively：
Sense primer is：5’-TGCGGCGGTAAAGGTTG-3’
Anti-sense primer is：5’-CGTCTGTGTAGAAACTGCGAATG-3’
C, result and judgement
Set during detection using the Plasmid DNA containing autotelic 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
Different in nature band, then it is special to show primer.According to occurring expected characteristic bands at 415bp, show in the feed containing dry
Lactobacillus paracasei does not contain Lactobacillus casei then on the contrary.
The Plasmid DNA of the amplified fragments containing target gene be by by the PCR product of Lactobacillus casei with
The positive clone molecule that pMD18T carriers 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
Goldview is dyed, and then carries out uv photography.
D, the extraction of RNA
(1) preparation of sample total serum IgE
Total serum IgE in Feed Sample is extracted using Trizol methods：Sterile working will be by the abundant feed 25g for crushing mixing
It is put into the sterilizing wide-mouth bottle of the sterile saline containing 225mL, (4 DEG C) are shaken with 100 revs/min of speed under cryogenic
1.5 hours are swung, are made into 10% uniform dilution.Sterile working carries out 10 times to uniform dilution prepared by previous step and is incremented by
Dilution selects the dilution after 3 or more suitable dilutions to add rapidly in feed dilution for extracting total serum IgE
The step of entering 1mLTrizol reagents, sample RNA, RNA extraction is extracted by Trizol kits (D9108A) specification is as follows：
1. adding in chloroform (1/5 volume of RNAiso Plus, 200L) into the homogenate lysate of above-mentioned steps, cover tightly
Centrifuge tube lid is shaken vigorously by hand for 15 seconds when vibration (chloroform low boiling point, volatile, careful centrifuge tube lid is answered to flick suddenly).It treats
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, i.e.,：It is colourless supernatant, intermediate
White egg white and with coloured lower floor's organic phase.Aspirate supernatant (usually taking 450L) be transferred to another new 1.5mL from
(white interlayer is never suctioned out in heart pipe)
4. isometric isopropanol (450L) is added in into supernatant, after the abundant mixing of the centrifuge tube that turns upside down, 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 is present with precipitation.
Careful supernatant discarding slowly adds in 75% ethyl alcohol 1mL (being sure not to touch precipitation), gently up and down along centrifugation tube wall
Washing centrifuge tube tube wall is overturned, ethyl alcohol is carefully discarded (in order to preferably control the salt in RNA after 12,000g 4 DEG C of centrifugation 5min
Ion concentration, should try one's best cleared ethyl alcohol).Reusable 75% ethyl alcohol 1mL is cleaned one time.
Drying at room temperature precipitates 5~10min (cannot centrifuge or heat drying, otherwise RNA will be difficult dissolving), adds in suitable
RNase-free water (enteron aisle usually takes 50L, and cell, bacterium take 20L) the dissolving precipitation of amount, can gently be blown if necessary with liquid-transfering gun
Precipitation is beaten, treats that RNA precipitate is completely dissolved and is placed on ice.
(2) Concentration Testing of RNA, dilution
1. the foundation of background：RNase-free water 2L are taken, are established on trace dna Protein Detection instrument NanoDrop 2000
Blank；The RNA concentration of RNase-free water is detected, is advisable in ± 5ng/L.
2. detect sample concentration：Detectable concentration difference is advisable tissue sample concentration with being no more than 50ng/L twice, is surveyed twice
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：
X L samples is taken to dilute, then without enzyme water additive amount：Without enzyme water volume Y=(original liquid concentration/600-1) * X (L), cell,
Bacteria samples need not generally 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, it is then that Sample storage is spare at -80 DEG C using the integrality of 1.0% agarose gel electrophoresis detection RNA；
E, post transcription cloning
10 μ L of reaction system：2.0μL 5×PrimeScript Buffer 2(for Real Time)、0.5μL
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, 85 DEG C add
Hot 5s finally by reverse transcription into 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 of PCR reaction systems：12.5μL 2×Premix Ex TaqTM, 10 μm of ol/L upstream and downstream primers
Each 0.5 μ L, cDNA template 2.0 μ L, dH2O9.5μL。
Quantitative fluorescent PCR response parameter：95 DEG C of pre-degeneration 3min；95 DEG C of denaturation 30s, 64 DEG C of annealing 30s, 72 DEG C extend
45s is cycled 40 times, and each sample is repeated 3 times, and is preserved under 4 DEG C of low temperature.
The Lactobacillus casei upstream and downstream specific primer is respectively：
Sense primer is：5’-TGCGGCGGTAAAGGTTG-3’
Anti-sense primer is：5’-CGTCTGTGTAGAAACTGCGAATG-3’
The preparation of outer standard items and the drafting of standard curve
The preparation of outer standard items：Extraction contains with the plasmid of the amplified fragments of the target gene of Lactobacillus casei type strain
DNA after ultraviolet specrophotometer measures A values, calculates copy number, carries out 10 times and be serially diluted, gradient dilution to 109-102It copies
The concentration of shellfish number/uL 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 obtains the standard curve of Lactobacillus casei for ordinate, as sample to be tested quantitative determination
Reference standard.
G, result and judgement
Using the DNA of sample to be tested cDNA and standard items as template, with the species-specific primer of Lactobacillus casei, with identical
System is carried out at the same time the fluorescent quantitative PCR of the target gene fragment of Lactobacillus casei, passes through standard curve and sample to be tested
Ct values the starting template amount of Lactobacillus casei in Feed Sample is obtained.
Embodiment 4：
Kit specific detection：
1) specificity verification of Lactobacillus casei qualitative detection：
The primer and method provided using embodiment 3, to lactobacillus acidophilus, lactobacillus plantarum, lactobacillus bulgaricus, dung
Enterococcus, enterococcus faecalis, bifidobacterium bifidum, animal bifidobacteria, Escherichia coli, the positive control (matter containing target gene
Grain be masterplate), Lactobacillus casei, the feed added with Lactobacillus casei；Negative control (aqua sterilisa) carries out PCR amplification.
The results show：Only swimming lane 1 (plasmid containing target gene is masterplate), 2 (Lactobacillus caseis), 3 (are added with cheese
The feed of lactobacillus) generate specificity 415bp amplified band, and other bacterial strains do not have, show provided by the invention
Primer only can specificity detection Lactobacillus casei.The amplification for having swimming lane 3 simultaneously understands that the sample of offer of the invention is pre-
Processing method can carry out separation and Extraction (Fig. 1) to Lactobacillus casei well.
2) Lactobacillus casei quantitatively detects specificity verification
The method provided using embodiment 3, with non-bacterium of the same race as reference strain, quantitatively detects Lactobacillus casei progress
Specificity verification.
Reference strain used has：Lactobacillus acidophilus；Lactobacillus plantarum；Lactobacillus bulgaricus；Enterococcus faecalis；Dung intestines ball
Bacterium；Bifidobacterium；Bifidobacterium adolescentis.
As a result judge：1. cycle threshold (Ct values)≤35 shows that PCR has the amplification of target dna in the process, can directly judge
For the positive；2. treat that sample genetic test Ct values between 32~40, should reform real-time fluorescent PCR amplification；It is outer after expanding again
Source gene C t values are still less than 40, and curve has apparent 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 values are more 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 Lactobacillus casei specific primer under conditions of, only exist
Under the conditions of template is Lactobacillus casei DNA, the amplification curve with apparent increased logarithmic phase can be just obtained, i.e. Lactobacillus casei is determined
Amount detection has specific (Fig. 2)
Embodiment 5：
Kit sensitivity detects：
Lactobacillus casei is cultivated into certain time in the medium, its A value is surveyed, its bacterium number is estimated, then by 10-1, 10-2,
10-3, 10-4, 10-5, 10-6, 10-7, 10-8Multiple dilution, the bacterium solution of last several gradients is taken to apply plate count, is repeated 3 times, takes
Its average value determines its real strain density.After each gradient bacterium solution respectively takes 3 pipes extraction RNA to be reversed to cDNA simultaneously, with to be measured
Sample cDNA is template, and Lactobacillus casei is carried out with the species-specific primer and method of the Lactobacillus casei described in embodiment 3
The fluorescent quantitative PCR (Fig. 3) of target gene fragment, and using plate count total bacteria count to numerical value as abscissa, fluorescence is fixed
The Ct values for measuring PCR reactions are ordinate, obtain the standard curve (Fig. 4) of bacterium number and Ct values.
The result shows that：Corresponding total bacteria count is the Monitoring lower-cut of Lactobacillus casei when Ct values are 32, as 8.5 ×
103Cfu, that is, the lowest detection standard of the fluorescent PCR detection of primer provided by the invention.
Embodiment 6：
In the feed without Lactobacillus casei, addition final concentration of 4.63 ± 0.21 × 108The cheese breast of cfu/g feeds
(plate count bacterium number is 4.8 × 10 to bacillus8Cfu/g feeds, 4.4 × 108Cfu/g feeds, 4.7 × 108Cfu/g feeds), it utilizes
The method of the present invention is detected, obtained real-time quantitative PCR detection Ct values for 21.66 ± 0.26 (Ct values respectively 21.47,
21.95th, 21.55), the standard curve y=-3.3252x+50.537 (R established using Lactobacillus casei standard items2=
0.9951), obtain in feed be Lactobacillus casei concentration be 4.89 ± 0.27 × 108(theoretical bacterium number is respectively cfu/g feeds
5.5×108Cfu/g feeds, 4.0 × 108Cfu/g feeds, 5.2 × 108Cfu/g feeds), through 17.0 statistical analyses of SPSS, put down
Plate is counted obtains two groups of data differences not significantly (P=0.604) with the method for the present invention, i.e. sample pretreating method of the invention
With reference to selected target gene, testing result has accuracy.
SEQUENCE LISTING
<110>Wuhan Polytechnic University
<120>Fast qualitative, immue quantitative detection reagent box and detection method for the Lactobacillus casei added in feed
And application
<130>Fast qualitative, immue quantitative detection reagent box and detection method for the Lactobacillus casei added in feed
And application
<160> 2
<170> PatentIn version 3.1
<210> 1
<211> 17
<212> DNA
<213>Artificial sequence
<400> 1
tgcggcggta aaggttg 17
<210> 2
<211> 23
<212> DNA
<213>Artificial sequence
<400> 2
cgtctgtgta gaaactgcga atg 23
Claims (5)
1. fast qualitative, immue quantitative detection reagent box for the Lactobacillus casei added in feed, including primer：5’-
TGCGGCGGTAAAGGTTG-3 ', 5 '-CGTCTGTGTAGAAACTGCGAATG-3 ', the application method of the kit, including
The preprocess method of Feed Sample, this method include：Sterile physiological salt by the feed 25g and 225mL that fully crush mixing
Water mixes, and shakes 1-2h with 100 revs/min of speed at 4 DEG C, is made into 10% uniform dilution；10 are carried out to uniform dilution
Incremental dilution again selects the dilution factor of 2 or more, as needed, extraction bacterial genomes DNA or RNA.
2. application of the kit described in claim 1 in the fast qualitative of Lactobacillus casei.
3. application of the kit described in claim 1 in Lactobacillus casei quantitatively detects.
4. application of the kit described in claim 1 in the qualitative and quantitative detection of Lactobacillus casei.
5. application of the kit described in claim 1 in Lactobacillus casei detection reagent is prepared.
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| CN104450941A (en) * | 2014-12-24 | 2015-03-25 | 光明乳业股份有限公司 | Method for detecting lactobacillus casei strain and kit and primer pair thereof |
| CN104531873A (en) * | 2014-12-24 | 2015-04-22 | 光明乳业股份有限公司 | Method for detecting lactobacillus casei strain and kit and primer pair used by method |
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| CN104450941A (en) * | 2014-12-24 | 2015-03-25 | 光明乳业股份有限公司 | Method for detecting lactobacillus casei strain and kit and primer pair thereof |
| CN104531873A (en) * | 2014-12-24 | 2015-04-22 | 光明乳业股份有限公司 | Method for detecting lactobacillus casei strain and kit and primer pair used by method |
Non-Patent Citations (1)
| Title |
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| 单核细胞增生李斯特菌PCR检测方法与试剂盒的建立;张丹丹;《中国优秀硕士学位论文全文数据库工程科技Ⅰ辑》;20130715;摘要，29页2.1.5节 * |
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