CN105925717A - Method and kit for detecting A1 and A2 beta-casein milk cows - Google Patents
Method and kit for detecting A1 and A2 beta-casein milk cows Download PDFInfo
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Abstract
The invention discloses a method and kit for detecting A1 and A2 Holstein cows, belonging to the technical field of molecular biology detection. The method comprises the following steps: designing special primers by using DNA of a cow to be detected as a template, carrying out PCR (polymerase chain reaction) amplification reaction, carrying out EcoT22I enzyme digestion on the amplification segment, and carrying out agarose gel electrophoresis detection, wherein the cow is an A1 cow if the length of the DNA segment subjected to gel detection is 434bp, the cow is an A1/A2 hybrid cow if the length of the DNA segment subjected to enzyme digestion is 434bp, 385bp and 49bp, and the cow is an A2 cow if the length of the DNA segment subjected to enzyme digestion is 385bp and 49bp. The detection method is convenient to operate, has the advantages of low cost and high accuracy, and can be used for screening A2 milk cows in the early state, thereby reducing the economic loss in cattle raising industry.
Description
Technical field
The invention belongs to technical field of molecular biological detection, be specifically related to a kind of detect A1 and A2 type beta-casein milch cow
Method and test kit thereof.
Background technology
Milk quality is the life of development of dairy industry, affects the sustainable health development of milk industry.Lactoprotein is the master constituting milk quality
Wanting material base, the lactoprotein in milk is based on casein and lactalbumin.Casein accounts for the 80% of milk protein, including
Alpha s1, alpha s2, beta, kappa tetra-type.Wherein β casein accounts for the 30% of total protein, is aminoacid
Important sources, research report beta-casein has A1, and A2, A3, A4, B, C, D, E, F, H1, H2, I, G are common
13 kinds of genetic variation types (Kami ski etc., 2007).A1 and A2 type beta-casein is in milch cow colony modal two kinds,
Its difference is that beta-casein gene there occurs a base change, thus causes relevant position aminoacid to be become group ammonia by proline
Acid.Research finds, just because of the amino acid whose change of said one, result in milk and creates difference during human digestive.
A1 type milk in digestion or the fresh milk course of processing some enzyme can at its histidine specific for hydrolysis, thus formed a kind of by seven
The peptide fragment of aminoacid composition, is referred to as β-hydrolyzed casein (BCM-7), and A2 type β casein aminoacid herein is proline, no
Therefore BCM-7 can not be formed by specific for hydrolysis.It has been found that BCM-7 can enter blood circulation through gastrointestinal wall,
Affect digestive system and immunocyte, may be relevant with some diseases, such as type Ⅰ diabetes mellitus, respiratory dysfunction, digestive system
Disease, immunologic function disorder and sudden infant death syndrome etc..Recently, latest domestic achievement in research shows, a lot of people couple
The cognitive presence mistaken ideas of lactose intolerance, the reason in fact causing their drink milk to occur as soon as intestinal reaction is possible as intestinal
Inflammatory reaction produced by the road A1 protein to containing in ordinary milk and cause.At present, the whole world only New Zealand sets up
The company of one production and selling A2 brand milk product of having the right, its all of A2 milk is all to be verified by DNA sequencing.
In November, 2013, A2 Co., Ltd. releases its A2Platinum platinum baby formula milk powder series to Chinese Consumer's first
Product, is also actively expanding other markets in North America and Asia.It may be said that the appearance of A2 brand is that scientific research is to business development
One time successful is broken through.
Along with publicity and the report of A1, A2 milk relevant knowledge, and food safety increasingly receives publicity, and A2 milk will be more
How favored by consumer.Therefore, it is necessary to provide a kind of effective differentiation A1 type and the side of A2 type beta-casein milch cow
Method.Chinese patent CN1052919839A discloses a kind of method based on enzyme cutting method detection beta-casein gene type, the method
It is that the base sequence according to beta-casein designs the primer that can amplify TaqI restriction enzyme site, utilizes primer to be detected
Milch cow genomic DNA carries out PCR amplification, produces a TaqI enzyme in the A1 type beta-casein gene fragment amplified
Cut site, utilize restricted enzyme TaqI that amplified production is carried out enzyme action, sentence according to the result of agarose gel electrophoresis
Disconnected, the amplified production containing A1 type beta-casein gene will produce the fragment of a 218bp and a 35bp after enzyme action
Small fragment.Although the method can realize the detection of beta-casein gene type, but when the method swims primer in design, wound
The mutational site making enzyme action is positioned at last base of this primer, from the agarose gel electrophoresis figure of this patent it can be seen that enzyme
With the presence of other electrophoretic bands many after cutting, the most mixed and disorderly, it is understood that there may be the length of small fragment after non-characteristic amplification phenomenon, and enzyme action
Degree only 35bp, the intersegmental length difference of sheet is the least, and these problems the most easily cause false positive or false-negative testing result occur.
Accordingly, it would be desirable to position, amplification condition and enzyme action system for primer creativeness restriction enzyme site do corresponding improvement, fast to meet
The needs of speed detection A1 and A2 type beta-casein milch cow.
Summary of the invention
For above-mentioned prior art, the present inventor, through performing creative labour and substantial amounts of test, has obtained can be used in detecting A1
With method and the test kit thereof of A2 type beta-casein milch cow, described detection method and the high specificity of test kit thereof, highly sensitive,
A1 and A2 type beta-casein milch cow can be detected fast and accurately.Thus provide following invention:
One aspect of the present invention relates to a kind of primer for detecting A1 and A2 type beta-casein milch cow, the sequence of described primer
Arrange as follows:
Beta-caseinF:5'-CCTTTGCCCAGACACAGTCTCTAGTCTATCCCTTCCCTGG GCCCATG
C-3'(SEQ ID NO.1)；
Beta-caseinR:5'-CTCCTGGTACAGCAGAAAGGCCTGAATGGGCATATCTC-3 ' (SEQ ID
NO.2)。
Use above-mentioned primer that genes of interest is expanded, if the 49th of pcr amplified fragment the is CC, for A1 type cattle, PCR
The sequence of amplified fragments is as shown in SEQ ID NO.3；If the 49th of pcr amplified fragment the is AA, for A2 type cattle, PCR
The sequence of amplified fragments is as shown in SEQ ID NO.4；If the 49th C and A base all exists in amplified fragments sequence, it is then
A1 and A2 heterozygosis cattle.
The application in the reagent or test kit of preparation detection A1 and A2 type beta-casein milch cow of the above-mentioned primer is also the present invention
Protection domain.
The design of primers thinking of the present invention is: based on beta-casein gene (Ensembl reference sequences
ENSBTAT00000003409) c.245C > A sudden change, and utilize PCR-RFLP technology at forward primer (beta-caseinF)
On create a mutational site, design obtains expanding the primer producing EcoT22I restriction enzyme site.
It should be noted that the design of primer is except usual design of primers annealing temperature to be considered, G/C content etc. in the present invention
Aspect, more to consider two key factors: the selection of the position, mutational site that (1) forward primer is created；(2) anti-after enzyme action
Answer the size of product fragment；If the fragment of the product after enzyme action is too small, then when carrying out detected through gel electrophoresis, bar interband shows
Show or cannot be distinguished by display false positive or false-negative testing result easily occur, but amplified production is also unsuitable long unintelligible,
Long, the efficiency of amplification can be affected.
The present invention is directed to forward primer and have selected different mutational sites, and the length of amplified production fragment after enzyme action, devise
Multipair primer, determines that after overtesting is verified repeatedly the present invention is above-mentioned listed for detecting A1 and A2 type beta-casein milk
The primer of cattle is optimal primer, it is possible to take into account the needs of the accuracy of detection, specificity and amplification efficiency.Other primers are then difficult to
Realize taking into account of detection accuracy and amplification efficiency.
Another aspect of the present invention relates to a kind of test kit for detecting A1 and A2 type beta-casein milch cow, and it comprises this
Bright above-mentioned primer.
In mentioned reagent box, also comprise Taq Master Mix, EcoT22I enzyme, Buffer buffer and DNA Marker.
Described Taq Master Mix is by Taq DNA Polymerase (0.05units/ μ l), MgCl2 (4mM) and dNTPs
(0.4mM) constitute.
In a specific embodiment of the present invention, described test kit comprises:
Above-mentioned primer (10 μm ol/L)；2×Taq Master Mix；10 × H Buffer buffer；EcoT22I enzyme；Aquesterilisa；
2000bp DNA Marker。
Another aspect of the invention relates to the method for detection A1 and the A2 type beta-casein milch cow of a kind of non-diagnostic purpose, including
Use above-mentioned primer that the genes of interest of testing sample is carried out the step of PCR amplification, pcr amplification product is carried out endonuclease reaction
Step, and the fragment after enzyme action is carried out the step of detected through gel electrophoresis.
Described PCR amplification employing 25 μ l reaction systems, consisting of of reaction system: template DNA (100ng/ μ l) 1 μ l, on
Trip primer beta-caseinF (10 μm ol/L), each 0.5 μ l of downstream primer beta-caseinR (10 μm ol/L), 2 × Taq Master
Mix 12.5 μ l, aquesterilisa 10.5 μ l.
The reaction condition of described PCR amplification is: 94 DEG C of denaturations 4min；Then 94 DEG C of degeneration 30s, 67 DEG C of annealing 30s, 72 DEG C
Extending 10s, this walks totally 35 circulations；72 DEG C keep 5min.
Described endonuclease reaction uses 20 μ l endonuclease reaction systems, consisting of of reaction system: pcr amplification product 10 μ l；10×H
Buffer buffer 2 μ l；EcoT22I enzyme 1 μ l；Aquesterilisa 7 μ l.
The reaction condition of described endonuclease reaction is: 37 DEG C of digestion 60min.
The step of described detected through gel electrophoresis is particularly as follows: by the agarose gel electrophoresis that fragment mass concentration is 3% after enzyme action.
The cattle to be measured of a length of 434bp of detected through gel electrophoresis enzyme action DNA fragmentation is A1 type cattle；Fragment is 434bp, 385bp and 49
Bp is A1 and A2 heterozygous cattle；The detection cattle of 385bp and 49bp is A2 type cattle.
Beneficial effects of the present invention:
The detection method operation of the present invention is quickly, accuracy is high and expense is low.Develop detection kit according to detection method simultaneously,
Making operation more standardized, this invention can detect A1 and A2 type Holstein cow in early days, eliminates the cowboying that A1 type cattle causes
Industry is lost, and has a extensive future, it is possible to produce considerable economic benefit and good social benefit in the molecular genetic breeding of cattle.
Accompanying drawing explanation
1% agarose gel electrophoresis figure of Fig. 1: pcr amplification product；
Fig. 2: utilize beta-caseinF and beta-caseinR to carry out 3% agarose gel electrophoresis figure of typing detection；In figure 1:
A2 type；2:A1 type；3:A1 and A2 heterozygous；Maker:2000bp DNA Marker；
Fig. 3: utilize the result figure of beta-caseinF-1 and beta-caseinR sensitivity tests to carry out 3% agarose of typing detection
Gel electrophoresis figure；1:A2 type in figure；2:A1 type；3:A1 and A2 heterozygous；Maker:2000bp DNA Marker.
Detailed description of the invention
The present invention is further illustrated in conjunction with the embodiments, it should explanation, the description below merely to explain the present invention,
Its content is not defined.The experimental technique of unreceipted actual conditions in following embodiment, generally according to normal condition, or
According to the condition proposed by reagent manufacturer；Material used in following embodiment, reagent etc., if no special instructions,
Obtain from commercial channels.
Embodiment 1: the method for detection A1 and A2 type beta-casein milch cow builds
1. design of primers
Based on beta-casein gene (Ensembl reference sequences ENSBTAT00000003409) c.245C > A sudden change, and
Creating a mutational site on forward primer, design is for the primer containing mutational site, thus creates EcoT22I enzyme action position
Point.Separately design out multipair primer, used the primer designed that genes of interest carries out PCR amplification respectively, and amplification is produced
Thing carries out EcoT22I enzyme action and detected through gel electrophoresis checking；Specific as follows:
(1) from Holstein cow blood, seminal fluid or hair follicle, the STb gene of cattle to be measured is extracted as genes of interest；
(2) use the primer of design that genes of interest is carried out PCR amplification；
PCR amplification uses 25 μ l reaction systems, consisting of of reaction system: template DNA (100ng/ μ l) 1 μ l, upstream is drawn
Thing beta-caseinF (10 μm ol/L), each 0.5 μ l of downstream primer beta-caseinR (10 μm ol/L), 2 × Taq Master Mix
12.5 μ l, aquesterilisa 10.5 μ l.
The reaction condition of PCR amplification is: 94 DEG C of denaturations 4min；Then 94 DEG C of degeneration 30s, 67 DEG C of annealing 30s, 72 DEG C are prolonged
Stretching 10s, this walks totally 35 circulations；72 DEG C keep 5min.
(3) pcr amplification product is carried out endonuclease reaction；
Endonuclease reaction uses 20 μ l endonuclease reaction systems, consisting of of reaction system: pcr amplification product 10 μ l；10×H Buffer
Buffer 2 μ l；EcoT22I enzyme 1 μ l；Aquesterilisa 7 μ l.
The reaction condition of endonuclease reaction is: 37 DEG C of digestion 60min.
(4) fragment after endonuclease reaction is carried out detected through gel electrophoresis；
The step of detected through gel electrophoresis is particularly as follows: by the agarose gel electrophoresis that fragment mass concentration is 3% after enzyme action.
Selecting two groups of representational primers, its sequence is as follows:
Beta-caseinF:5'-CCTTTGCCCAGACACAGTCTCTAGTCTATCCCTTCCCTGG GCCCATG
C-3'；
Beta-caseinR:5'-CTCCTGGTACAGCAGAAAGGCCTGAATGGGCATATCTC-3 '.
Beta-caseinF-1:5'-CAGTCTCTAGTCTATCCCTTCCCTGGGCCCATG-3'；
Beta-caseinR:5'-CTCCTGGTACAGCAGAAAGGCCTGAATGGGCATATCTC-3 '.
Expanding with above-mentioned two groups of primers, the gel electrophoresis figure after amplified production enzyme action is distinguished the most as shown in Figures 2 and 3, by scheming
It can be seen that after the amplified production enzyme action of different primers, the Detection results of its gel electrophoresis is different, with beta-caseinF-1 and
The product that beta-caseinR obtains as primer amplification, when carrying out detected through gel electrophoresis after enzyme action, band shows unintelligible, and
Little band cannot be carried out distinguishing display (Fig. 3), and this is probably the position, enzyme action mutational site that forward primer beta-caseinF-1 creates
Last base or enzyme action small fragment the shortest (only 35bp) in this primer is difficult to differentiate between causing.It is directed to this, we
Design beta-caseinF forward primer, the penultimate base at this primer creates enzyme action mutational site, and expands upstream
Primer length, and optimize detection scheme accordingly.
Therefore, the following sequence primer sequence as detection A1 and A2 type beta-casein milch cow is determined through assay optimization screening.
Beta-caseinF:5'-CCTTTGCCCAGACACAGTCTCTAGTCTATCCCTTCCCTGG GCCCATG
C-3'(SEQ ID NO.1)；
Beta-caseinR:5'-CTCCTGGTACAGCAGAAAGGCCTGAATGGGCATATCTC-3 ' (SEQ ID
NO.2)。
2. the optimization of detection method
The optimization of 2.1PCR reaction condition
In the PCR reaction system of 25 μ l, on impact reaction several conditions be optimized, including annealing temperature (55～75 DEG C),
Primer concentration (1～20 μm ol/L) and response time and period etc., observe its impact on testing result, determine and most preferably move back
The reaction conditions such as fire temperature, primer concentration, response time and period.
Result shows: optimum PCR reaction condition is: primer concentration is 10 μm ol/L, 94 DEG C of denaturations 4min；Then 94 DEG C
Degeneration 30s, 67 DEG C of annealing 30s, 72 DEG C extend 10s, and this walks totally 35 circulations；72 DEG C keep 5min.Anti-with above-mentioned PCR
The amplification efficiency answering condition to carry out reacting is the highest.
The optimization of 2.2 endonuclease reaction conditions
In the endonuclease reaction system of 20 μ l, the temperature (25-45 DEG C) to endonuclease reaction, digestion time (30-120min) is carried out
Optimize.
Result shows: optimum endonuclease reaction condition is 37 DEG C of digestion 60min.
Embodiment 2: for detecting the test kit of A1 and A2 type beta-casein milch cow
The composition of test kit includes: primer (10 μm ol/L)；2×Taq Master Mix；10 × H Buffer buffer；EcoT22
I enzyme；Aquesterilisa；2000bp DNA Marker.
Primer sets is that in embodiment 1, screening and optimizing obtains, and its sequence is:
Beta-caseinF:5'-CCTTTGCCCAGACACAGTCTCTAGTCTATCCCTTCCCTGG GCCCATG
C-3'(SEQ ID NO.1)；
Beta-caseinR:5'-CTCCTGGTACAGCAGAAAGGCCTGAATGGGCATATCTC-3 ' (SEQ ID
NO.2)。
Taq Master Mix is by Taq DNA Polymerase (0.05units/ μ l), MgCl2 (4mM) and dNTPs (0.4mM)
Constitute.
The detection method using this test kit to carry out Clinical detection is:
(1) from Holstein cow blood, seminal fluid or hair follicle, extract the STb gene of cattle to be measured；
(2) use the primer of design that genes of interest is carried out PCR amplification；
PCR amplification uses 25 μ l reaction systems, consisting of of reaction system: template DNA (100ng/ μ l) 1 μ l, upstream is drawn
Thing beta-caseinF (10 μm ol/L), each 0.5 μ l of downstream primer beta-caseinR (10 μm ol/L), 2 × Taq Master Mix
12.5 μ l, aquesterilisa 10.5 μ l.
The reaction condition of PCR amplification is: 94 DEG C of denaturations 4min；Then 94 DEG C of degeneration 30s, 67 DEG C of annealing 30s, 72 DEG C are prolonged
Stretching 10s, this walks totally 35 circulations；72 DEG C keep 5min.
(3) pcr amplification product is carried out endonuclease reaction；
Endonuclease reaction uses 20 μ l endonuclease reaction systems, consisting of of reaction system: pcr amplification product 10 μ l；10×H Buffer
Buffer 2 μ l；EcoT22I enzyme 1 μ l；Aquesterilisa 7 μ l.
The reaction condition of endonuclease reaction is: 37 DEG C of digestion 60min.
(4) fragment after endonuclease reaction is carried out detected through gel electrophoresis；
The step of detected through gel electrophoresis is particularly as follows: by the agarose gel electrophoresis that fragment mass concentration is 3% after enzyme action, gel
The cattle to be measured of a length of 434bp of electrophoresis detection enzyme action DNA fragmentation is A1 type cattle；Fragment is 434bp, 385bp and 49bp
For A1 and A2 heterozygous cattle；The detection cattle of 385bp and 49bp is A2 type cattle.
Embodiment 3: the specific detection of test kit
With the A1 of known type, A2, A3, A4 type beta-casein milch cow is detection object, uses the test kit of embodiment 2
Detecting, result is: A1, A2 type beta-casein milch cow can run out of the band of corresponding length in gel electrophoresis, and A3,
A4 type beta-casein milch cow cannot run out of the band of corresponding length.Show the primer sets in test kit of the present invention, PCR reaction system
With detection method, there is good specificity.
Embodiment 4: the repeatability detection of test kit
With the type beta-casein milch cow of A1 and A2 of known type for detection object, the test kit of the present invention is used to carry out many
Secondary repetition is tested, and the result that detection is repeated several times is all consistent, shows the test kit of the present invention and the good stability of detection method.
Embodiment 5: clinical sample detects
12 Holstein cow are detected by the test kit using the embodiment of the present invention 2.
According to above-mentioned detection A1 and the test kit of A2 type Holstein cow, following steps PCR are utilized to expand cattle beta-casein base
Because of fragment sequence, the fragment mass concentration of amplification is 1% agarose gel electrophoresis detection, and result is as shown in Figure 1.
(1) PCR reaction system: DNA (100ng/ μ l) 1 μ l, on, each 0.5 μ l of downstream primer (10 μm ol/L), 2 × Taq
Master Mix 12.5 μ l, aquesterilisa 10.5 μ l.
(2) PCR reaction condition: 94 DEG C of denaturations 4min；94 DEG C of degeneration 30s, 67 DEG C of annealing 30s, 72 DEG C extend 10s,
This walks totally 35 circulations；72 DEG C keep 5min.
(3) electrophoresis detection of amplified production: take 3 μ l PCR primer and carry out agarose gel electrophoresis detection.Detection display 12
The DNA sample of swimming lane is single band, and size is 434bp, consistent with purpose clip size.
The enzyme action typing detection of 3.PCR product
(1) 20 μ l enzyme action system: pcr amplification product 10 μ l；10 × H Buffer buffer 2 μ l；EcoT22I enzyme 1 μ l；
Aquesterilisa 7 μ l.37 DEG C of enzyme action 60min.
(2) electrophoresis detection: be that 3% agarose gel carries out electrophoresis detection to the 20 μ l altogether of the product after enzyme action by mass concentration；
Voltage 90V；Time 40min.
4. gene type and interpretation of result: the genotyping result of A1 type, A2 type and heterozygous cattle is as shown in table 1 below.
Altogether have detected 12 Holstein cow, after wherein having 2 cattle enzyme action, DNA fragmentation is 434bp, for A1 type cattle；3
Head DNA fragmentation be 434bp, 385bp and 49bp be A1 and A2 heterozygous cattle；The detection cattle of 7 385bp and 49bp
For A2 type cattle.
1. Ns of beta-casein genotyping result of table
All of sample standard deviation is through direct Sequencing detection checking, and result is completely the same with the testing result using test kit of the present invention,
Rate of accuracy reached 100%, illustrates that the test kit of the present invention and detection method thereof are accurate and effective, high specificity, and testing result is without false sun
Property and false negative.
Claims (10)
1. the primer being used for detecting A1 and A2 type beta-casein milch cow, it is characterised in that the sequence of described primer is:
Beta-caseinF:5'-CCTTTGCCCAGACACAGTCTCTAGTCTATCCCTTCCCTGG GCCCATG
C-3'；
Beta-caseinR:5'-CTCCTGGTACAGCAGAAAGGCCTGAATGGGCATATCTC-3 '.
2. the answering in the reagent or test kit of preparation detection A1 and A2 type beta-casein milch cow of the primer described in claim 1
With.
3., for detecting a test kit for A1 and A2 type beta-casein milch cow, it comprises the primer described in claim 1.
4. test kit as claimed in claim 3, it is characterised in that in described test kit, also comprise Taq Master Mix,
EcoT22 I enzyme, Buffer buffer and DNA Marker.
5. test kit as claimed in claim 4, it is characterised in that described Taq Master Mix is by Taq DNA
Polymerase(0.05units/μl)、MgCl2(4mM) constitute with dNTPs (0.4mM).
6. the method for detection A1 and the A2 type beta-casein milch cow of a non-diagnostic purpose, it is characterised in that include using power
Profit requires that the primer described in 1 carries out the step of PCR amplification to the genes of interest of testing sample, and pcr amplification product is carried out enzyme
Cut the step of reaction, and the fragment after enzyme action is carried out the step of detected through gel electrophoresis.
7. method as claimed in claim 6, it is characterised in that described PCR amplification uses 25 μ l reaction systems, reactant
System consists of: template DNA 1 μ l, forward primer beta-caseinF, each 0.5 μ l of downstream primer beta-caseinR, 2 × Taq
Master Mix 12.5 μ l, aquesterilisa 10.5 μ l.
8. method as claimed in claim 6, it is characterised in that the reaction condition of described PCR amplification is: 94 DEG C of denaturations
4min；Then 94 DEG C of degeneration 30s, 67 DEG C of annealing 30s, 72 DEG C extend 10s, totally 35 circulations；72 DEG C keep 5min.
9. method as claimed in claim 6, it is characterised in that described endonuclease reaction uses 20 μ l endonuclease reaction systems, reaction
Consisting of of system: pcr amplification product 10 μ l；10 × H Buffer buffer 2 μ l；EcoT22I enzyme 1 μ l；Aquesterilisa 7 μ l.
10. method as claimed in claim 6, it is characterised in that the step of described detected through gel electrophoresis is: after enzyme action
Fragment mass concentration is the agarose gel electrophoresis of 3%.The a length of 434bp's of detected through gel electrophoresis enzyme action DNA fragmentation is to be measured
Cattle is A1 type cattle；Fragment be 434bp, 385bp and 49bp be A1 and A2 heterozygous cattle；The inspection of 385bp and 49bp
Surveying cattle is A2 type cattle.
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| CN107287292A (en) * | 2017-06-07 | 2017-10-24 | 山东省农业科学院奶牛研究中心 | The one group of primer special and its kit of milk cow β casein different variants types are detected simultaneously |
| CN108157293A (en) * | 2018-02-07 | 2018-06-15 | 山东省农业科学院奶牛研究中心 | A kind of breeding method for simplifying selection high productivity energy A2A2 homozygous genotype milk cows based on pedigree information |
| CN108823321A (en) * | 2018-06-25 | 2018-11-16 | 北京向中生物技术有限公司 | A kind of the HRM detection method and primer of beta-casein gene parting |
| CN109283239A (en) * | 2018-10-22 | 2019-01-29 | 山东省农业科学院奶牛研究中心 | A kind of different beta-casein variant type method in detection cow's milk |
| KR102715907B1 (en) | 2023-09-15 | 2024-10-14 | 주식회사 미코바이오메드 | COMPOSITION AND METHOD FOR DETECTING β-CASEIN A1 TYPE GENE AND β-CASEIN A2 TYPE GENE IN MILK |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107287292A (en) * | 2017-06-07 | 2017-10-24 | 山东省农业科学院奶牛研究中心 | The one group of primer special and its kit of milk cow β casein different variants types are detected simultaneously |
| CN107287292B (en) * | 2017-06-07 | 2019-11-26 | 山东省农业科学院奶牛研究中心 | The one group of primer special and its kit of milk cattle beta-casein different variants type are detected simultaneously |
| CN108157293A (en) * | 2018-02-07 | 2018-06-15 | 山东省农业科学院奶牛研究中心 | A kind of breeding method for simplifying selection high productivity energy A2A2 homozygous genotype milk cows based on pedigree information |
| WO2019153823A1 (en) * | 2018-02-07 | 2019-08-15 | 山东省农业科学院奶牛研究中心 | Pedigree information simplification-based breeding method for selecting high-production performance a2a2 homozygous genotype dairy cattle |
| CN108157293B (en) * | 2018-02-07 | 2020-03-20 | 山东省农业科学院奶牛研究中心 | Breeding method for simplified selection of high-production-performance A2A2 homozygous genotype cows based on pedigree information |
| CN108823321A (en) * | 2018-06-25 | 2018-11-16 | 北京向中生物技术有限公司 | A kind of the HRM detection method and primer of beta-casein gene parting |
| CN109283239A (en) * | 2018-10-22 | 2019-01-29 | 山东省农业科学院奶牛研究中心 | A kind of different beta-casein variant type method in detection cow's milk |
| CN109283239B (en) * | 2018-10-22 | 2022-01-04 | 山东省农业科学院奶牛研究中心 | Method for detecting different beta-casein variant types in cow milk |
| KR102715907B1 (en) | 2023-09-15 | 2024-10-14 | 주식회사 미코바이오메드 | COMPOSITION AND METHOD FOR DETECTING β-CASEIN A1 TYPE GENE AND β-CASEIN A2 TYPE GENE IN MILK |
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