CN109402265A - Method based on KASP technology detection beta lactoglobulin genotype - Google Patents
Method based on KASP technology detection beta lactoglobulin genotype Download PDFInfo
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Abstract
The present invention relates to a kind of methods based on KASP technology detection beta lactoglobulin genotype, include the following steps: to prepare DNA sample；Prepare PCR amplification system；Carry out PCR amplification；Mix primer in PCR amplification system includes three kinds of KASP primers: the unmutated gene primer in upstream, upstream mutated gene primer and downstream primer, also, HEX fluorescence labels sequence and FAM mutation fluorescence labels sequence are respectively designed in the unmutated gene primer in upstream and upstream mutated gene primer；According to the fluorescence intensity of DNA sample after PCR amplification, Genotyping is carried out.The present invention realizes comprehensive screening to two kinds of common variants of BLG gene in milk cow, the resource in the fields such as genetic test and technical advantage can be made full use of to cultivate high-quality kind of ox, excavate milk cow characteristic germ plasm resource, suitable milk cow is selected for pasture, improve milk production trait, the technical bottleneck that related fields is solved from provenance is of great significance to the sound development of milk cow breeding industry.
Description
Technical field
The present invention relates to molecular biology and Biotechnology in Genetic Breeding fields, and in particular to one kind detects β-based on KASP technology
The method of lactoglobulin gene type.
Background technique
Beta lactoglobulin (β-lactoglobulin, β-LG) is the peculiar albumen synthesized by galactophore epithelial cell, is generally deposited
It is in the milk of a variety of mammals such as reinder, pig, horse, sheep, cat and dog.The lactoprotein of milk is mainly by casein and whey
Albumen composition, accounts for about the 80% and 20% of milk total protein respectively, and β-LG is the main component of lactalbumin, accounts for lactalbumin
50%, account for the 12% of total protein.
The main purpose of research milk protein gene polymorphism is that the auxiliary choosing of ox breeding is applied to as genetic marker
In selecting.The milk production trait of ox is the quantitative character controlled by multidigit point, genetic force be generally it is medium on the lower side, it is affected by environment
Greatly, and milk protein gene site is then one kind in all influential gene locis of multipair milk production trait.It looks forward to the future, from β-cream
Globulin present Research can be seen that application potential of the molecular genetics methods in cattle breeding is very big, have wide development
Prospect.
Research with the introducing and development of Protocols in Molecular Biology and nucleic acid, largely based on beta lactoglobulin gene
Report show the gene pleiomorphism it is significant, coding β-LG albumen gene be referred to as BLG gene, on the locus find A, B,
C, D, E, F, G, H and W9 kind Isoforms.Beta lactoglobulin is primarily present two kinds of genetic variants of A, B in cow's milk, causes this at two
The site of base replacement is located at the 3rd and the 4th exon of BLG gene.Research has shown that different variants to milk cow cow's milk
Component, property and yield have a certain impact, it was found that β-Lg gene pleiomorphism also has an impact to Holstein cow Reproduction.B
Variant is preponderant genotype, plays positive improving effect.
The method of detection beta-casein gene type has digestion, sequencing, fluorescent quantitative PCR technique etc., but above method at present
It is bothersome, laborious and at high cost.
Summary of the invention
There is solution the shortcomings of the prior art, the present invention provides one kind to detect beta lactoglobulin base based on KASP technology
Because of the method for type, include the following steps:
Step S1: preparation DNA sample；
Step S2: PCR amplification system is prepared, wherein the interior DNA sample including 50% volume fraction of the amplification system,
The mix primer of 1.2-1.6% volume fraction and the KASP mixed liquor of surplus；
Step S3: PCR amplification is carried out；
Wherein, the mix primer added in step S2 include three kinds of KASP primers, respectively the unmutated gene primer in upstream,
Upstream mutated gene primer and downstream primer, also, in the unmutated gene primer in upstream and upstream mutated gene primer respectively
Design has HEX fluorescence labels sequence and FAM fluorescence labels sequence, with by the fluorescence intensity according to DNA sample after PCR amplification,
Carry out Genotyping.
Wherein, in the step S2, added mix primer includes two groups, is used to detect mutational site at two respectively
Base type, also, be directed to different mutational sites, the primer of design is as follows:
(1) when base type on the site LG-118 to be detected, primer includes: upstream mutated gene primer
CACCCAGGCACTGGCAGG, the unmutated gene primer CCACCCAGGCACTGGCAGA in upstream and downstream primer
AGTACCTGCTCTTCTGCATGGAGAA；
(2) when base type on the site LG-64 to be detected, primer includes: upstream mutated gene primer
ATGATCTTCTTCTGAGCACACTCAC, the unmutated gene primer AATGATCTTCTTCTGAGCACACTCAT in upstream and under
Swim primer GAAAGCAGCTGTCTTTCAGGGAGAA.
It wherein,, should if corresponding FAM fluorescence intensity only occurs in DNA after amplification when detecting the base type in corresponding site
Two chains are mutated on site；If corresponding HEX fluorescence intensity only occurs in DNA after amplification, two chains are unmutated on the site；
If two kinds of mixing fluorescence intensities occurs in DNA after amplification, two one, chain mutation on the site, one unmutated.
Wherein, in the step S3, the step of PCR amplification, includes:
Step S31: when temperature is 90-95 degrees Celsius hot activation 14-16 minutes, 90-95 degrees Celsius of time variation 18-22
Second, it anneals at 65-50 degrees Celsius and extends 55-65 second, carry out 10 and recycle；
Step S32: annealing when temperature is 90-95 degrees Celsius time variation 18-22 seconds, 60-50 degrees Celsius and extends 55-65
Second, carry out 24-28 circulation；
Step S33: annealing when temperature is 90-95 degrees Celsius time variation 18-22 seconds, 60-50 degrees Celsius and extends 55-65
Second, carry out 3 circulations.
Wherein, in the step S3, the step of PCR amplification, includes:
Step S31: temperature be 94 degrees Celsius when hot activation 15 minutes, 94 degrees Celsius time variation 20 seconds, 61-55 degrees Celsius
When anneal and extend 60 seconds, carry out 10 circulation；
Step S32: annealing when temperature is 94 degrees Celsius time variation 20 seconds, 55 degrees Celsius and extend 60 seconds, carries out 26
Circulation；
Step S33: annealing when temperature is 94 degrees Celsius time variation 20 seconds, 57 degrees Celsius and extend 60 seconds, carries out 3 and follows
Ring.
Wherein, in the step S1, the extracting method of DNA sample includes:
Step S11: blood-material is pre-processed；
Step S12: blood-material is successively digested by Porteinase K solution and buffer GB, keeps solution system clear
Clearly；
Step S13: addition buffer solution B D mixes well；
Step S14: solution is placed in adsorption column CG2, and waste liquid is abandoned in room temperature centrifugation；
Step S15: being added buffer GDB, and waste liquid is abandoned in room temperature centrifugation；
Step S16: being added buffer PWB, and room temperature centrifugation is abandoned waste liquid, is repeated at least once more；
Step S17: the adsorption column CG2 finally obtained is placed in collecting pipe, and waste liquid is abandoned in room temperature centrifugation；
Step S18: elution adsorption column CG2 it is quantitative to carry out DNA；
Step S19: the integrality of DNA sample is detected by agarose gel electrophoresis.
Wherein, in the step S11, carrying out pretreated step to blood-material includes: that 1- is added into blood sample
The cell pyrolysis liquid CL of 2.5 times of volumes, is handled using Syrup-homogenizing instrument, is centrifuged later, and supernatant is abandoned, and leaves nucleus precipitating,
Buffer GS is added into nucleus precipitating, oscillation is mixed to thorough；
Wherein, if without blood clot in blood-material, the volume ratio of the buffer GS of the volume and addition of blood-material is
5:1, if there is blood clot in blood-material, the volume ratio of the buffer GS of the volume and addition of blood-material is 5:2.
Wherein, in the step S12, if blood-material is without clot, the body of Porteinase K solution and blood-material
Ratio is accumulated as 1:50, the volume ratio of buffer GB and blood-material is 1:5；If blood-material has clot, Porteinase K is molten
The volume ratio of liquid and blood-material is 1:25, and the volume ratio of buffer GB and blood-material is 2:5.
Wherein, in the step S13, the volume of added buffer GB in added buffer solution B D and step S12
Than between 1:1-2:1；
In the step S15, the volume ratio of added buffer GB is situated between in added buffer GDB and step S12
In 2:1-3:1；
In the step S16, the volume ratio of added buffer GB is situated between in added buffer PWB and step S12
In 2:1-4:1；
Also, the step S14, into step S16, for the speed of centrifugation between 10000-14000rpm, centrifugation time is equal
Between 20-40 seconds；
In the step S17, the speed of centrifugation is between 10000-14000rpm, and centrifugation time is between 1.5-2.5 points.
Wherein, in the step S18, the elution process of adsorption column CG2 includes:
Step S18a: elution buffer TB, the volume that elution buffer TB is added, with step is vacantly added dropwise to adsorption column CG2
The volume ratio of added buffer GB is between 1:4-1:1 in rapid S12；
Step S18b: being placed at room temperature for two minutes, and waste liquid is abandoned in room temperature centrifugation, centrifugal speed between 10000-14000rpm, from
The heart time is between 1.5-2.5 points.
Method provided by the invention based on KASP technology detection beta lactoglobulin genotype, realizes to BLG in milk cow
Comprehensive screening of two kinds of common variants of gene can make full use of the resource in the fields such as genetic test to cultivate with technical advantage high-quality
Kind ox, excavates milk cow characteristic germ plasm resource, selects suitable milk cow for pasture, improves milk production trait, from the related neck of provenance solution
The technical bottleneck in domain is of great significance to the sound development of milk cow breeding industry.
Detailed description of the invention
Fig. 1: method one genotyping result figure of the invention based on KASP technology detection beta lactoglobulin genotype.
Specific embodiment
In order to have further understanding to technical solution of the present invention and beneficial effect, it is described in detail with reference to the accompanying drawing
Technical solution of the present invention and its beneficial effect of generation.
The present invention is directed to be screened by Genotyping to common variants in three in CSN3 (κ-CN gene) comprehensively,
To provide reference for scientific seed selection and selective pairing.
One, the preparation of DNA sample
In the present invention, using the blood of milk cow as extraction, the material of preparation DNA sample.
1, blood-material pre-processes:
(1) it if blood takes 1000ul blood to be tested without blood clot, need to be handled with cell pyrolysis liquid CL, specifically
Step: being added the cell pyrolysis liquid CL of 1-2.5 times of volume, be mixed by inversion in sample, 10,000rpm (11,500g) are centrifuged 1min,
Supernatant is sucked, nucleus precipitating (if cracking is not thorough, it is primary to repeat above step) is left, to the cell being collected by centrifugation
In core precipitating plus 200ul buffer GS, oscillation are mixed to thorough.
(2) if blood has blood clot, 500ul blood clot is taken to be tested, need to be handled with cell pyrolysis liquid CL, specifically
Step: in sample be added 1-2.5 times of volume cell pyrolysis liquid CL, handled using Syrup-homogenizing instrument, carried out after being disposed from
The heart 10,000rpm (11,500g), 1min, sucks supernatant, leaves nucleus precipitating and (if cracking is not thorough, repeats the above step
It is rapid primary), into the nucleus precipitating being collected by centrifugation plus 200ul buffer GS, oscillation are mixed to thorough.
If blood is to freeze state, it need to thaw in 37 DEG C of water-baths, forbid thaw at RT.
It if it is new blood and has been layered, heparin tube gentle inversion need to have been mixed, then carried out taking blood step.
Accordingly, with respect to common kit, the present invention as desired by the additive amount of blood (no blood clot), by
200ul is promoted to 1000ul (blood is without grumeleuse) or 500ul (blood has blood clot) and carries out pre-treatment, makes the base finally extracted
Because group DNA concentration is in 100ng/ul or more, detection accuracy is increased.
2, into the blood sample pre-processed, 20ul Porteinase K is added, mixes well.
3,200ul buffer GB is added, is sufficiently mixed by inversion, 56 DEG C of water-bath 10min are mixed by inversion for several times therebetween, make molten
Liquid, which sufficiently digests, becomes limpid.
If solution does not become limpid thoroughly, need to extend pyrolysis time until solution is limpid.
4,350ul buffer solution B D is added after being placed at room temperature for 2-5min, is sufficiently mixed by inversion.This step is likely to occur cotton-shaped heavy
It forms sediment, but without dispelling.
5, upper step acquired solution and flocculent deposit are added in adsorption column CG2 (adsorption column is put into collecting pipe), room temperature from
The heart, 12,000rpm (13,400g), 30sec abandon waste liquid.
6,500ul buffer GDB, room temperature centrifugation, 12,000rpm (13,400g), 30sec, abandoning waste liquid is added.
7,600ul rinsing liquid PWB, room temperature centrifugation, 12,000rpm (13,400g), 30sec, abandoning waste liquid is added.
8,600ul rinsing liquid PWB, room temperature centrifugation, 12,000rpm (13,400g), 30sec, abandoning waste liquid is added.
9, CG2 adsorption column is put back in collecting pipe, room temperature centrifugation, 12,000rpm (13,400g), 2min abandons waste liquid.
10, adsorption column CG2 pipe lid is opened, is placed at room temperature for several minutes, thoroughly to dry remaining rinsing liquid in adsorbent material.
11, adsorption column CG2 is put into a clean 1.5ml EP pipe, is washed to the hanging 50-200ul that is added dropwise in adsorbed film center
De- buffer TB is placed at room temperature for 2min, room temperature centrifugation, 12,000rpm (13,400g), 2min.
For the yield for increasing genomic DNA, obtained solution can be rejoined in adsorption column CG2, be placed at room temperature for 2min,
Room temperature centrifugation, 12,000rpm (13,400g), 2min.
12, it is quantitative to carry out gDNA, if at once not quantitative, sample is frozen.
13, agarose gel electrophoresis detects gDNA integrality.
Two, the design of primer
Beta lactoglobulin genotyping method of the invention is absorbed in and detects tri- kinds of genotype of A, B and E therein, and three
Kind genotype is related to six kinds of different milk cow genomes, also, is related to the catastrophe point everywhere on CSN3.
Table 1 is four group primer sequences and each primer amplification band institute of the present invention for the mutation point design of different location
Corresponding base type, table 2 are in the present invention, and six kinds of different milk cow genomes are corresponding on four different mutational sites
Base type.
Table 1: primer of the present invention for different mutation point designs
Table 2: corresponding base type on the different mutational sites that the present invention is measured
| Genotype | β-LG-64 | β-LG-118 |
| AA | A | T |
| AB | GA | CT |
| BB | G | C |
Three, PCR amplification
In the present invention, the PCR system for amplification is 5 μ L, including 2.5 μ L DNA, 2.5 Μ l KASP Master Mix
(LGC Genomics, Hoddeston, UK, KASP mixed liquor), 0.07 μ L mix primer.To avoid the erroneous judgement to test result,
Blank control, DNA profiling ddH are set2O (distilled water) is replaced.The hot activation when temperature is 94 DEG C of the condition of PCR amplification
It anneals when 15min, 94 DEG C of time variation 20s, 61-55 DEG C and extends 60s, carries out 10 circulations；When 94 DEG C of time variation 20s, 55 DEG C
Annealing and extension 60s, 26 circulations are carried out；It anneals when 94 DEG C of time variation 20s, 57 DEG C and extends 60s, carries out 3 circulations.
Four, genotyping result
As described above, in table 1 of the invention, every group of PCR amplification primer includes three kinds of amplified bands: the unmutated gene in upstream
Primer, upstream mutated gene primer and downstream primer, also, in the unmutated gene primer in upstream and upstream mutated gene primer
It is respectively designed with HEX fluorescence labels sequence and FAM fluorescence labels sequence；After PCR amplification, difference can be presented in different DNA samples
Fluorescence intensity.By taking LG-118 catastrophe point as an example, incorporated by reference to shown in Fig. 1, it is assumed that certain milk cow (milk cow at this time unmutated here
Genotype is AA), then the base type at the position is T, only will appear the corresponding expansion of the unmutated gene primer in upstream after DNA cloning
Increase band, that is, HEX, which is presented, in the DNA finally expanded corresponds to fluorescence intensity, the i.e. color (navy blue) in the upper left corner Fig. 1；Assuming that certain milk
Ox has the mutation of chain (genotype of milk cow is AB at this time) here, then the base type of a chain is C at the position, and another
For T, can occur the unmutated gene primer in upstream and the corresponding amplified band of upstream mutated gene primer simultaneously after DNA cloning, that is,
The DNA presentation HEX finally expanded corresponds to fluorescence intensity and FAM corresponds to the secondary colour of fluorescence intensity, i.e. iridescent among Fig. 1；
Assuming that two chains are mutated (genotype of milk cow is BB at this time) to certain milk cow here, then base type is C at the position, and DNA expands
It only will appear mutated gene primer corresponding amplified band in upstream after increasing, to correspond to fluorescence strong that is, FAM is presented in the DNA that finally expands
The color of degree, the i.e. color (red) in the lower right corner Fig. 1.
It, can also by the fluorescence intensity of the corresponding amplification of amplified band corresponding at another place mutational site LG-64
To judge the genotype of milk cow, principle is similar as above, and the present invention does not add tired state.
It is so-called " Proteinase K solution " in the present invention, refer to Proteinase K Solution；So-called cell pyrolysis liquid CL,
Buffer GDB, buffer GB, buffer GS, buffer PWB, buffer solution B D, adsorption column CG2 and eluent TB, are selected from day
Root kit self-contained reagent.
Method provided by the invention based on KASP technology detection beta lactoglobulin genotype, realizes to BLG in milk cow
Comprehensive screening of two kinds of common variants of gene can make full use of the resource in the fields such as genetic test to cultivate with technical advantage high-quality
Kind ox, excavates milk cow characteristic germ plasm resource, selects suitable milk cow for pasture, improves milk production trait, from the related neck of provenance solution
The technical bottleneck in domain is of great significance to the sound development of milk cow breeding industry.
The breeding plan that the genotyping result provided through the invention carries out genetic improvement, which can be convenient, quickly to be implemented, and can
To be generalized in the analysis of gene loci, can be answered according to its genotype combination production performance and physical efficiency character and heredity estimation
For matching system and breeding practice, as long as finding ideal type male animal in group, DNA typing combination artificial insemination, embryo are utilized
The methods of tire transplanting and breeding estimation, so that it may so that the ideal genotype with excellent genetic background is fixed rapidly, compared to more sharp
With the method for genetic engineering, the present invention does not have any risk, easy to be reliable, in production practice, to raising merit
Operability is stronger for genetic progress.
Although the present invention is illustrated using above-mentioned preferred embodiment, the protection model that however, it is not to limit the invention
It encloses, anyone skilled in the art are not departing within the spirit and scope of the present invention, and opposite above-described embodiment carries out various changes
It is dynamic still to belong to the range that the present invention is protected with modification, therefore protection scope of the present invention subjects to the definition of the claims.
Claims (10)
1. a kind of method based on KASP technology detection beta lactoglobulin genotype, which comprises the steps of:
Step S1: preparation DNA sample；
Step S2: PCR amplification system is prepared, wherein DNA sample, 1.2- in the amplification system including 50% volume fraction
The mix primer of 1.6% volume fraction and the KASP mixed liquor of surplus；
Step S3: PCR amplification is carried out；
Wherein, the mix primer added in step S2 includes three kinds of KASP primers, the respectively unmutated gene primer in upstream, upstream
Mutated gene primer and downstream primer, also, separately designed in the unmutated gene primer in upstream and upstream mutated gene primer
There are HEX fluorescence labels sequence and FAM fluorescence labels sequence, to carry out by the fluorescence intensity according to DNA sample after PCR amplification
Genotyping.
2. the method as described in claim 1 based on KASP technology detection beta lactoglobulin genotype, which is characterized in that described
In step S2, added mix primer includes two groups, is used to detect the base type in mutational site at two respectively, also,
For different mutational sites, the primer of design is as follows:
(1) when base type on the site LG-118 to be detected, primer includes: upstream mutated gene primer
CACCCAGGCACTGGCAGG, the unmutated gene primer CCACCCAGGCACTGGCAGA in upstream and downstream primer
AGTACCTGCTCTTCTGCATGGAGAA；
(2) when base type on the site LG-64 to be detected, primer includes: upstream mutated gene primer
ATGATCTTCTTCTGAGCACACTCAC, the unmutated gene primer AATGATCTTCTTCTGAGCACACTCAT in upstream and under
Swim primer GAAAGCAGCTGTCTTTCAGGGAGAA.
3. the method as claimed in claim 2 based on KASP technology detection beta lactoglobulin genotype, which is characterized in that detection
When base type in corresponding site, if corresponding FAM fluorescence intensity only occurs in DNA after amplification, two chains are dashed forward on the site
Become；If corresponding HEX fluorescence intensity only occurs in DNA after amplification, two chains are unmutated on the site；If DNA occurs after amplification
Two kinds of mixing fluorescence intensities, then two one, chain mutation on the site, one unmutated.
4. the method as described in claim 1 based on KASP technology detection beta lactoglobulin genotype, which is characterized in that described
In step S3, the step of PCR amplification, includes:
Step S31: temperature be 90-95 degrees Celsius when hot activation 14-16 minutes, 90-95 degrees Celsius time variation 18-22 seconds, 65-
It anneals and extends 55-65 seconds at 50 degrees Celsius, carry out 10 circulations；
Step S32: annealing when temperature is 90-95 degrees Celsius time variation 18-22 seconds, 60-50 degrees Celsius and extend 55-65 seconds,
Carry out 24-28 circulation；
Step S33: annealing when temperature is 90-95 degrees Celsius time variation 18-22 seconds, 60-50 degrees Celsius and extend 55-65 seconds,
Carry out 3 circulations.
5. the method as claimed in claim 4 based on KASP technology detection beta lactoglobulin genotype, which is characterized in that described
In step S3, the step of PCR amplification, includes:
Step S31: it is moved back when temperature being 94 degrees Celsius hot activation 15 minutes, 94 degrees Celsius time variation 20 seconds, 61-55 degrees Celsius
Fire and extension 60 seconds, carry out 10 circulations；
Step S32: annealing when temperature is 94 degrees Celsius time variation 20 seconds, 55 degrees Celsius and extend 60 seconds, carries out 26 circulations；
Step S33: annealing when temperature is 94 degrees Celsius time variation 20 seconds, 57 degrees Celsius and extend 60 seconds, carries out 3 circulations.
6. the method as described in claim 1 based on KASP technology detection beta lactoglobulin genotype, which is characterized in that described
In step S1, the extracting method of DNA sample includes:
Step S11: blood-material is pre-processed；
Step S12: blood-material is successively digested by Porteinase K solution and buffer GB, clarifies solution system；
Step S13: addition buffer solution B D mixes well；
Step S14: solution is placed in adsorption column CG2, and waste liquid is abandoned in room temperature centrifugation；
Step S15: being added buffer GDB, and waste liquid is abandoned in room temperature centrifugation；
Step S16: being added buffer PWB, and room temperature centrifugation is abandoned waste liquid, is repeated at least once more；
Step S17: the adsorption column CG2 finally obtained is placed in collecting pipe, and waste liquid is abandoned in room temperature centrifugation；
Step S18: elution adsorption column CG2 it is quantitative to carry out DNA；
Step S19: the integrality of DNA sample is detected by agarose gel electrophoresis.
7. the method as claimed in claim 6 based on KASP technology detection beta lactoglobulin genotype, which is characterized in that described
In step S11, the cell that pretreated step includes: the 1-2.5 times of volume of addition into blood sample is carried out to blood-material and is split
Liquid CL is solved, is handled using Syrup-homogenizing instrument, is centrifuged later, supernatant is abandoned, leaves nucleus precipitating, is added into nucleus precipitating
Buffer GS, oscillation are mixed to thorough；
Wherein, if without blood clot in blood-material, the volume ratio of the buffer GS of the volume and addition of blood-material is 5:1,
If there is blood clot in blood-material, the volume ratio of the buffer GS of the volume and addition of blood-material is 5:2.
8. the method as claimed in claim 6 based on KASP technology detection beta lactoglobulin genotype, which is characterized in that described
In step S12, if blood-material, without clot, the volume ratio of Porteinase K solution and blood-material is 1:50, buffer
The volume ratio of GB and blood-material is 1:5；If blood-material has clot, the volume of Porteinase K solution and blood-material
Than for 1:25, the volume ratio of buffer GB and blood-material is 2:5.
9. the method as claimed in claim 6 based on KASP technology detection beta lactoglobulin genotype, which is characterized in that
In the step S13, the volume ratio of added buffer GB is between 1:1- in added buffer solution B D and step S12
2:1；
In the step S15, the volume ratio of added buffer GB is between 2 in added buffer GDB and step S12:
1-3:1；
In the step S16, the volume ratio of added buffer GB is between 2 in added buffer PWB and step S12:
1-4:1；
Also, the step S14 is into step S16, the speed of centrifugation between 10000-14000rpm, centrifugation time between
20-40 seconds；
In the step S17, the speed of centrifugation is between 10000-14000rpm, and centrifugation time is between 1.5-2.5 points.
10. the method as claimed in claim 6 based on KASP technology detection beta lactoglobulin genotype, which is characterized in that institute
It states in step S18, the elution process of adsorption column CG2 includes:
Step S18a: elution buffer TB, the volume that elution buffer TB is added, with step S12 is vacantly added dropwise to adsorption column CG2
In added buffer GB volume ratio between 1:4-1:1；
Step S18b: being placed at room temperature for two minutes, and waste liquid is abandoned in room temperature centrifugation, and centrifugal speed is between 10000-14000rpm, when centrifugation
Between between 1.5-2.5 point.
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