CN109321639A - κ-casein genotype method is detected based on KASP technology - Google Patents
κ-casein genotype method is detected based on KASP technology Download PDFInfo
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Abstract
κ-casein genotype method is detected based on KASP technology the present invention relates to a kind of, includes 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: upstream mutated gene primer, the unmutated gene primer in upstream and downstream primer, and, in the unmutated gene primer of upstream mutated gene primer and upstream, one design has HEX fluorescence labels sequence, and another design has FAM fluorescence labels sequence；According to the fluorescence intensity of DNA sample after PCR amplification, Genotyping is carried out.The present invention realizes comprehensive screening to tri- kinds of common variants of CSN3 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 protein ratio, 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 κ-based on KASP technology
The method of casein genotype.
Background technique
κ-casein (kappa-casein, k-CN) be cow's milk glandular secretion a kind of phosphoprotein containing a small amount of phosphate it
One.Content of the κ-casein in cow's milk accounts for about the 12% of total casein, is the natural substrate of renin.And κ-CN is most heavy
One of casein wanted, it not only serve in terms of the stability of casein micelles it is vital, but also to Lactation of Dairy Cow
Performance, milk composition composition and cheese quality have a degree of influence.
Coding κ-casein gene is named as CSN gene, it has now been found that, there are many CSN bases of type in ox
Cause.The κ of bovine animals-casein gene CSN3 synthesizes protein and the stabilization of micellar casein plays an important role, in milk
It is of great significance in junket production, is the natural substrate of renin.There are 15 kinds of variants in ox κ-CN, it is non-synonymous by nucleotide
Replacement causes.Most common three genetic variants are A, B and E, they have difference, A in 136,148,155,168 amino acids
Four sites be respectively Thr, Asp, Ser, Ala, and B is Ile, Ala, Ser, Ala, E Thr, Asp, Gly, Ala.Accordingly
Nucleotide replacement is respectively ACC → ATC, GAT → GCT, AGC → GGC, GCA → GCG.
Many studies have shown that κ-CN gene may will affect the output of milk, butterfat production, Milk protein yield, the butterfat percnetage (F) of cow
With protein ratio (P), and the coagulating property and cheese yield of cow's milk are had a significant impact.Studies have shown that κ-CN is to butterfat percnetage
It has a significant impact.The milk butterfat percnetage with higher of the variant of-CNB containing κ, faster curdled milk speed and higher grumeleuse are hard
Degree.κ-CN protein genetic polymorphisms have a significant impact economic characters such as cheese making characteristic etc..Especially newborn rheology
Characteristic is learned, κ-CNB allele shows positive influence to casein number.Casein content is milk as cheesemaking
One important indicator of raw material.Therefore, screening is carried out to which the quality for improving dairy product is necessary to κ-CN gene.
With the development of molecular biotechnology, DNA analysis brings new vitality to cow's milk protein polymorphism research.Successively
Establish DNA level analysis lactoprotein genetic variant method, main method have PCR-SSCP, PCR-RFLP, direct Sequencing,
Allele specific pcr and genetic chip etc., due to first two method is easier to operate and to equipment requirement it is not high, application is more general
Time, rear three kinds of methods detection is accurate, but higher cost, is not suitable for large sample detection.
The country is more suitable for there has been no being associated with κ-casein gene polymorphism with cow's milk processing characteristics to filter out at present
The report of cheese production or the higher ox of protein yield only.
Summary of the invention
There is solution the shortcomings of the prior art, the present invention provides one kind to detect κ-casein gene based on KASP technology
The method of type, includes 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 upstream mutated gene primer, on
Unmutated gene primer and downstream primer are swum, also, in the unmutated gene primer of upstream mutated gene primer and upstream, one
Design has HEX fluorescence labels sequence, and another design has FAM fluorescence labels sequence, by according to DNA sample after PCR amplification
Fluorescence intensity, carry out Genotyping.
Wherein, in the step S2, added mix primer includes four groups, is used to detect mutational site everywhere respectively
Base type, also, be directed to different mutational sites, the primer of design is as follows:
(1) when base type on the site CSN3_136 to be detected, primer includes: the unmutated gene primer in upstream
GAGCCTACAAGTACACCTACCAC, upstream mutated gene primer GAGCCTACAAGTACACCTACCAT and downstream primer
GTAGCTACAGTGCTCTCTACTGCTT；
(2) when base type on the site CSN3_148 to be detected, primer includes: the unmutated gene primer in upstream
AGAGCACTGTAGCTACTCTAGAAGA, upstream mutated gene primer AGCACTGTAGCTACTCTAGAAGC and downstream primer
GTTGATCTCAGGTGGGCTCTCAATA；
(3) when base type on the site CSN3_155 to be detected, primer includes: the unmutated gene primer in upstream
GTGTTGATCTCAGGTGGGCT, upstream mutated gene primer GTGTTGATCTCAGGTGGGCC and downstream primer
GAGCACTGTAGCTACTCTAGAAGCTT；
(4) when base type on the site CSN3_168 to be detected, primer includes: upstream mutated gene primer
TGATGTCTCCTTAGAGTATTTAGACC, the unmutated gene primer CTTTGATGTCTCCTTAGAGTATTTAGACT in upstream with
And downstream primer TGAGATCAACACAGTCCAAGTTACTTCAA.
Wherein, when base type on (1) site CSN3_136 to be detected, if corresponding FAM only occur glimmering by DNA after amplification
Luminous intensity, then two chains are unmutated on the site；If corresponding HEX fluorescence intensity only occurs in DNA after amplification, two on the site
Chain is mutated；If two kinds of mixing fluorescence intensities occurs in DNA after amplification, two one, chain mutation on the site, one is not dashed forward
Become；
(2) when base type on the site CSN3_148 to be detected, if corresponding FAM fluorescence only occur strong by DNA after amplification
Degree, then two chains are unmutated on the site；If only there are corresponding HEX fluorescence intensity, two chains on the site in DNA after amplification
It is mutated；If two kinds of mixing fluorescence intensities occurs in DNA after amplification, two one, chain mutation on the site, one unmutated；
(3) when base type on the site CSN3_155 to be detected, if corresponding FAM fluorescence only occur strong by DNA after amplification
Degree, then two chains are unmutated on the site；If only there are corresponding HEX fluorescence intensity, two chains on the site in DNA after amplification
It is mutated；If two kinds of mixing fluorescence intensities occurs in DNA after amplification, two one, chain mutation on the site, one unmutated；
(4) when base type on the site CSN3_168 to be detected, if corresponding FAM fluorescence only occur strong by DNA after amplification
Degree, then two chains are mutated on the site；If corresponding HEX fluorescence intensity only occurs in DNA after amplification, two chains are equal on the site
It is unmutated；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.
It is provided by the invention that κ-casein genotype method is detected based on KASP technology, it realizes to CSN3 in milk cow
Comprehensive screening of (κ-CN gene) three kinds of common variants can make full use of the resource in the fields such as genetic test and technical advantage to train
High-quality kind of ox is educated, milk cow characteristic germ plasm resource is excavated, suitable milk cow is selected for pasture, improves protein ratio, solved from provenance
The technical bottleneck of related fields is of great significance to the sound development of milk cow breeding industry.
Detailed description of the invention
Fig. 1: of the invention that one genotyping result figure of κ-casein genotype method is detected based on KASP technology.
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
κ of the invention-casein genotype classifying method is absorbed in and detects tri- kinds of genotype of A, B and E therein, and three kinds
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 | CSN3_136 | CSN3_148 | CSN3_155 | CSN3_168 |
| AA | C | A | A | A |
| AB | CT | AC | A | GA |
| AE | C | A | AG | A |
| BB | T | C | A | G |
| BE | CT | AC | AG | GA |
| EE | C | A | G | A |
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: upstream mutated gene draws
Object, the unmutated gene primer in upstream and downstream primer, also, the unmutated gene primer of upstream mutated gene primer and upstream
In, a design has HEX fluorescence labels sequence, and another design has FAM fluorescence labels sequence；After PCR amplification, different DNA
Different fluorescence intensities can be presented in sample.By taking CSN3_136 catastrophe point as an example, incorporated by reference to shown in Fig. 1, it is assumed that certain milk cow is here
Unmutated (genotype of milk cow may be AA, AE or EE at this time), then the base type at the position is C, can only be gone out after DNA cloning
The existing corresponding amplified band of the unmutated gene primer in upstream, that is, FAM, which is presented, in the DNA finally expanded corresponds to fluorescence intensity, i.e. Fig. 1
The color (red) in the lower right corner；Assuming that certain milk cow have here the mutation of chain (at this time the genotype of milk cow may for AB or
BE), then the base type of a chain is C at the position, and another is T, can occur upstream mutated gene primer simultaneously after DNA cloning
And the corresponding amplified band of the unmutated gene primer in upstream, that is, HEX, which is presented, in the DNA finally expanded corresponds to fluorescence intensity and FAM pairs
Answer the secondary colour of fluorescence intensity, i.e. iridescent among Fig. 1；Assuming that two chains are mutated (milk cow at this time to certain milk cow here
Genotype is BB), then base type is T at the position, only will appear the corresponding amplification of upstream mutated gene primer after DNA cloning
Band, that is, the color that HEX corresponds to fluorescence intensity, the i.e. color (navy blue) in the upper left corner Fig. 1 is presented in the DNA finally expanded.
In conjunction with the amplification of other catastrophe points, can comprehensive descision milk cow genotype.
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.
It is provided by the invention that κ-casein genotype method is detected based on KASP technology, it realizes to CSN3 in milk cow
Comprehensive screening of (κ-CN gene) three kinds of common variants can make full use of the resource in the fields such as genetic test and technical advantage to train
High-quality kind of ox is educated, milk cow characteristic germ plasm resource is excavated, suitable milk cow is selected for pasture, improves protein ratio, solved from provenance
The technical bottleneck of related fields is of great significance to the sound development of milk cow breeding industry.
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 detect κ-casein genotype method based on KASP technology, 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, respectively upstream mutated gene primer, upstream not
Mutated gene primer and downstream primer, also, in the unmutated gene primer of upstream mutated gene primer and upstream in, one sets
In respect of HEX fluorescence labels sequence, another design has FAM fluorescence labels sequence, by according to DNA sample after PCR amplification
Fluorescence intensity carries out Genotyping.
2. detecting κ-casein genotype method based on KASP technology as described in claim 1, which is characterized in that the step
In rapid S2, added mix primer includes four groups, is used to detect the base type in mutational site everywhere, also, needle respectively
To different mutational sites, the primer of design is as follows:
(1) when base type on the site CSN3_136 to be detected, primer includes: the unmutated gene primer in upstream
GAGCCTACAAGTACACCTACCAC, upstream mutated gene primer GAGCCTACAAGTACACCTACCAT and downstream primer
GTAGCTACAGTGCTCTCTACTGCTT；
(2) when base type on the site CSN3_148 to be detected, primer includes: the unmutated gene primer in upstream
AGAGCACTGTAGCTACTCTAGAAGA, upstream mutated gene primer AGCACTGTAGCTACTCTAGAAGC and downstream primer
GTTGATCTCAGGTGGGCTCTCAATA；
(3) when base type on the site CSN3_155 to be detected, primer includes: the unmutated gene primer in upstream
GTGTTGATCTCAGGTGGGCT, upstream mutated gene primer GTGTTGATCTCAGGTGGGCC and downstream primer
GAGCACTGTAGCTACTCTAGAAGCTT；
(4) when base type on the site CSN3_168 to be detected, primer includes: upstream mutated gene primer
TGATGTCTCCTTAGAGTATTTAGACC, the unmutated gene primer CTTTGATGTCTCCTTAGAGTATTTAGACT in upstream with
And downstream primer TGAGATCAACACAGTCCAAGTTACTTCAA.
3. detecting κ-casein genotype method based on KASP technology as claimed in claim 2, which is characterized in that
(1) when base type on the site CSN3_136 to be detected, if only there is corresponding FAM fluorescence intensity in DNA after amplification,
Two chains are unmutated on the site；If corresponding HEX fluorescence intensity only occurs in DNA after amplification, two chains are prominent on the site
Become；If two kinds of mixing fluorescence intensities occurs in DNA after amplification, two one, chain mutation on the site, one unmutated；
(2) when base type on the site CSN3_148 to be detected, if only there is corresponding FAM fluorescence intensity in DNA after amplification,
Two chains are unmutated on the site；If corresponding HEX fluorescence intensity only occurs in DNA after amplification, two chains are prominent on the site
Become；If two kinds of mixing fluorescence intensities occurs in DNA after amplification, two one, chain mutation on the site, one unmutated；
(3) when base type on the site CSN3_155 to be detected, if only there is corresponding FAM fluorescence intensity in DNA after amplification,
Two chains are unmutated on the site；If corresponding HEX fluorescence intensity only occurs in DNA after amplification, two chains are prominent on the site
Become；If two kinds of mixing fluorescence intensities occurs in DNA after amplification, two one, chain mutation on the site, one unmutated；
(4) when base type on the site CSN3_168 to be detected, if only there is corresponding FAM fluorescence intensity in DNA after amplification,
Two chains are mutated on the site；If corresponding HEX fluorescence intensity only occurs in DNA after amplification, two chains are not dashed forward on the site
Become；If two kinds of mixing fluorescence intensities occurs in DNA after amplification, two one, chain mutation on the site, one unmutated.
4. detecting κ-casein genotype method based on KASP technology as described in claim 1, which is characterized in that the step
In rapid 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. detecting κ-casein genotype method based on KASP technology as claimed in claim 4, which is characterized in that the step
In rapid 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. detecting κ-casein genotype method based on KASP technology as described in claim 1, which is characterized in that the step
In rapid 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. detecting κ-casein genotype method based on KASP technology as claimed in claim 6, which is characterized in that the step
In rapid S11, carrying out pretreated step to blood-material includes: that the cell cracking of 1-2.5 times of volume is added into blood sample
Liquid CL, is handled using Syrup-homogenizing instrument, is centrifuged later, and supernatant is abandoned, and leaves nucleus precipitating, and into nucleus precipitating, addition is slow
Fliud flushing 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. detecting κ-casein genotype method based on KASP technology as claimed in claim 6, which is characterized in that the step
In rapid S12, if blood-material, without clot, the volume ratio of Porteinase K solution and blood-material is 1:50, buffer GB
Volume ratio with blood-material is 1:5；If blood-material has clot, the volume ratio of Porteinase K solution and blood-material
For 1:25, the volume ratio of buffer GB and blood-material is 2:5.
9. detecting κ-casein genotype method based on KASP technology as claimed in claim 6, 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. detecting κ-casein genotype method based on KASP technology as claimed in claim 6, which is characterized in that described
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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