CN108034728A - SNP marker for detecting lung cancer susceptibility combines, primer combines and kit - Google Patents

Abstract

The present invention relates to for detecting, the SNP marker of lung cancer susceptibility combines, primer combines and kit.SNP marker combination of the present invention includes rs1048943, rs1051730, rs13181, rs1799793 and rs25487.SNP marker of the present invention combination can system, comprehensively reflect lung cancer susceptibility, be particularly suitable for using in Chinese population, contribute to early detection lung cancer population, so as to targetedly better people's living environment and habits and customs, lower lung cancer morbidity rate.

Description

SNP marker for detecting lung cancer susceptibility combines, primer combines and kit

Technical field

The present invention relates to field of molecular marker, in particular to combined for detecting the SNP marker of lung cancer susceptibility, Primer combines and kit.

Background technology

Lung cancer is having become the most common malignant tumour in the whole world and the first tumor mortality reason in recent years, in China, Lung cancer is the primary cancer cause of the death.Gene, environment and life style three are the causes of disease for causing lung cancer, wherein gene rise Effect it is mostly important.Counted according to lung cancer morbidity age level, in the patients with lung cancer of 50 years old or so, it is by gene to have 27% Caused, it is as caused by gene and smoking joint effect to have 42%, and it is as caused by smoking and environmental factor to have 27%, is had 4% is as caused by other reasons.Up to the present, the key for improving lung cancer curative effect is early detection, early diagnosis, morning Phase treats, but is constrained to current diagnosis and treatment means, and middle and advanced stage is belonged to when 70% patients with lung cancer is made a definite diagnosis.Therefore send out as early as possible Existing lung cancer Susceptible population simultaneously implements precautionary measures, is of great significance to preventing the generation of lung cancer, improving therapeutic effect.

In recent years, many single nucleotide polymorphism (Single Nucleotide Polymorphisms, SNP) are found It is related to the neurological susceptibility of disease, and for the detection of disease susceptibility, wherein being no lack of and the relevant SNP marker of the neurological susceptibility of lung cancer With corresponding detection technique.But it is special that the combination of the SNP site of existing detection lung cancer susceptibility lacks systemic, comprehensive and crowd It is ineffective during the lung cancer susceptibility of the opposite sex, in the detection compatriots.

In addition, existing SNP marker is mainly detected by direct sequencing and Taqman sonde methods.Wherein directly survey High labor cost, the detection cycle length of sequence method, the main agents BigDye used in sequencing is import, and consumables cost is high, PCR The kit cost of product purification is also higher, cause direct sequencing is of high cost, detecting step is complicated, detection cycle length, very Difficulty realizes the detection of high throughput sample.And TaqMan fluorescence probe methods need expensive and do not possess the double-color probe of versatility, together When requirement to sample quality it is high, in addition to sample is without degraded, it is also necessary to which concentration is consistent as far as possible.

Therefore, this area urgently obtain one group can system and the SNP marker combination of reflection lung cancer susceptibility comprehensively, and pin Build vertical quick, detection method efficiently and accurately jointly to the mark group.

In view of this, it is special to propose the present invention.

The content of the invention

The SNP marker that the first object of the present invention is to provide for detecting lung cancer susceptibility combines, the SNP marker group Close can system, comprehensively reflect lung cancer susceptibility, be particularly suitable for Chinese population use, contribute to early detection lung cancer people Group, so as to targetedly better people's living environment and habits and customs, lowers lung cancer morbidity rate.

The second object of the present invention is to provide to be prepared for detecting lung for detecting the reagent of foregoing SNP marker combination Application in the reagent of cancer neurological susceptibility.

The third object of the present invention is to provide the KASP primers combination for detecting foregoing SNP marker combination, described to draw Thing combination quickly, accurately and efficiently can carry out parting by KASP technologies to foregoing SNP marker.

The fourth object of the present invention is to provide the kit for detecting foregoing SNP marker combination, the kit bag Include foregoing primer and other auxiliary detection reagents, can it is quick by KASP technologies, accurately and efficiently to foregoing SNP marker into Row parting.

In order to realize the above-mentioned purpose of the present invention, spy uses following technical scheme：

SNP marker for detecting lung cancer susceptibility combines, SNP marker combination include rs1048943, Rs1051730, rs13181, rs1799793 and rs25487.

The present invention accounts in terms of carcinogenic substance metabolism and DNA reparations and people's group specificity, finally selects The SNP marker group of rs1048943, rs1051730, rs13181, rs1799793 and rs25487 as detection lung cancer susceptibility Close.Lung cancer main reason is covered in SNP marker combination of the present invention, can reflect the neurological susceptibility of the lung cancer of various different origins, Therefore, it is possible to the neurological susceptibility of the various lung cancer of complete detection, meanwhile, more occurred frequently in the Chinese population and and lung cancer of above-mentioned SNP marker The neurological susceptibility degree of association is high, therefore, particularly suitable for detecting the neurological susceptibility of Chinese population lung cancer.

Wherein, the corresponding genes of SNP marker rs1048943 are cytochrome pathways 1A1 (CYP1A1).CYP1A1 Gene code aryl hydrocarbon hydroxylase, is I phase metabolic enzymes, participates in benzoin and other polycyclic arene compounds (contain in smoke from cigarette Have) etc. precarcinogen metabolism activation, the electrophilic glycol epoxides of formation combined with DNA, induces DNA mutation or activating cancer Gene, causes the vicious transformation of cell.SNP marker rs1048943, is the conversion for A → G that the 7th extron 131bp occurs, Base mutation causes amino acid to be changed into valine (Val) by isoleucine (Ile), ferroheme of this variation positioned at zymoprotein Land, the enzymatic activity of activity ratio's normal gene expression of mutator expression is high, and the ability of Val/Val activation poisonous substances is most By force.In Chinese population, G is risk allele, and compared with AA genotype, GG genotype increases the onset risk of lung cancer, OR It is worth for 1.61 (95%CI=1.24-2.08).

SNP marker rs13181 and rs1799793 correspond to Excision Repair Cross-Complementing Group (Excision Repair Cross-Complementing Group 2,ERCC2).A kind of DNA helicase of evolution conservative of ERCC2 gene codes, is core An important ring in thuja acid excision reparation approach.DNA damage position is once found that the helicase activity is enabled it to by specific protein The double-spiral structure of DNA is enough untied, the DNA oligonucleotide fragments of damage are removed or remove, particularly to causing due to smoking Nucleotide damage.

The change of SNP marker rs13181, T → G cause encoded amino acid to be sported by 751Lys (lysine) 751Gln (glutamine), reduces the DNA repair abilities of Nucleotide Sequence Analysis path, suffers from Lung Cancer Risk with individual and increases It is closely related.The ability that individuals of the Gln/Gln than Lys/Lys repairs pyrimidine dimer is low by 50%.In Chinese population, G is wind Dangerous allele, GG genotype adds the onset risk of lung cancer compared with TT genotype, and OR values are 3.19 (95%CI1.01- 10.07).Polymorphic site rs1799793, G → A polymorphism causes encoded amino acid to be mutated by 312Asp (aspartic acid) For 312Asn (asparagine), in Chinese population, A is risk allele, and AA genotype is relative to GG genotype, OR (10.33 95%CI=1.29-82.50).

The corresponding genes of SNP marker rs25487 repair Cross-complementing Gene 1 (XRCC1) for x-ray, are a kind of important DNA Revision points, play weight in DNA single-strand breaks caused by a series of oxidants (including tobacco component) and base injury repair Act on, participating in DNA single-strand breaks and base injury repair, the polymorphism in XRCC1 gene coding regions mononucleotide site can change Become the relative risk that body suffers from lung cancer.

The polymorphism in SNP marker rs25487 sites is G → A, causes corresponding amino acid replacement Arg → Gln, causes The injury repair parasthenia of XRCC1, as Gln variation allele number increases can cause the increase of lung cancer relative risk, A For risk allele.

It is CHRNA3 (nicotinic acetylcholine receptor that SNP marker rs1051730, which corresponds to gene, Alpha 3), be nAChR member, coding produce one kind can with nicotine with reference to and play its function Albumen, participate in the material such as nicotine and tobacco metabolism and internal signal transduction.The SNP marker rs1051730 of the gene is more State property is the base mutation of C → T, and in Asian, the risk that TT genotype carriers suffer from lung cancer increases by 1.51 times, has with lung cancer There are stronger correlation (p=5 × 10^-9)。

The invention further relates to the reagent for detecting foregoing SNP marker combination to prepare the examination for being used for detecting lung cancer susceptibility Application in agent.

In some specific embodiments, the reagent is used for ApoE gene, PCR sequencing PCR, Taqman and visits One or more in the skill of handling needles, Single strand conformation polymorphism, denaturing gradient gel electrophoresis or high-resolution solubility curve method；

Preferably, the reagent is used for ApoE gene；

It is highly preferred that the ApoE gene is competitive ApoE gene.

The invention further relates to the KASP primers combination for detecting foregoing SNP marker combination, the primer combination includes：

The primer 1~3 of rs1048943 is detected, the primer 1 is from 5 ' ends to 3 ' ends by sequence label 1 and such as SEQ ID NO：Nucleotide sequence shown in 6 forms, and the primer 2 is from 5 ' ends to 3 ' ends by sequence label 2 and such as SEQ ID NO：Core shown in 7 Nucleotide sequence forms, and the primer 3 is from 5 ' ends to 3 ' ends by SEQ ID NO：Nucleotide sequence shown in 8 forms；

The primer 4~6 of rs1051730 is detected, the primer 4 is from 5 ' ends to 3 ' ends by sequence label 1 and such as SEQ ID NO：Nucleotide sequence shown in 9 forms, and the primer 5 is from 5 ' ends to 3 ' ends by sequence label 2 and such as SEQ ID NO：Core shown in 10 Nucleotide sequence forms, and the primer 6 is from 5 ' ends to 3 ' ends by SEQ ID NO：Nucleotide sequence shown in 11 forms；

The primer 7~9 of rs13181 is detected, the primer 7 is from 5 ' ends to 3 ' ends by sequence label 1 and such as SEQ ID NO： Nucleotide sequence shown in 12 forms, and the primer 8 is from 5 ' ends to 3 ' ends by sequence label 2 and such as SEQ ID NO：Nucleosides shown in 13 Acid sequence forms, and the primer 9 is from 5 ' ends to 3 ' ends by SEQ ID NO：Nucleotide sequence shown in 14 forms；

The primer 10~12 of rs1799793 is detected, the primer 10 is from 5 ' ends to 3 ' ends by sequence label 1 and such as SEQ ID NO：Nucleotide sequence shown in 15 forms, and the primer 11 is from 5 ' ends to 3 ' ends by sequence label 2 and such as SEQ ID NO：Shown in 16 Nucleotide sequence forms, and the primer 12 is from 5 ' ends to 3 ' ends by SEQ ID NO：Nucleotide sequence shown in 17 forms；

With the primer 13~15 of detection rs25487, the primer 13 is from 5 ' ends to 3 ' ends by sequence label 1 and such as SEQ ID NO：Nucleotide sequence shown in 18 forms, and the primer 14 is from 5 ' ends to 3 ' ends by sequence label 2 and such as SEQ ID NO：Shown in 19 Nucleotide sequence forms, and the primer 15 is from 5 ' ends to 3 ' ends by SEQ ID NO：Nucleotide sequence shown in 20 forms.

Primer of the present invention combination be applicant for KASP (Kompetitive Allele Specific PCR, KASP) specially exploitation is used for the primer for detecting foregoing SNP marker to detection method.Wherein it is used for the primer for detecting each SNP site Including two sense primers with sequence label and the anti-sense primer without sequence label, it is with being connected to fluorophor It is used cooperatively with the fluorescence probe and quenching probes of quenching group, the polymorphic of the above-mentioned SNP marker of KASP technology for detection can be passed through Property, have that accuracy rate is high, flexibility is strong, without expensive Two Colour Fluorescence label probe, of less demanding to sample and can realize big The advantages of flux detects.

It is well known that primer sequence is the key of PCR reactions.Compared with general PCR, requirement of the KASP technologies to primer is more Height, due to the addition of universal primer, its factor needed to be considered is more, the difficulty of design also bigger, it is necessary to consider upper and lower Primer, amplified fragments are swum in G/C content, T_mWhether the matching of value, free energy etc., can form primer dimerization each other Whether body itself can form hairpin structure, and there is greatly uncertainty in terms of amplification efficiency.KASP primers at present Design depend on the Kraken of LCC companies^TMSoftware is designed, but the design principle of the software and software are in itself It is in confidential state, and there are larger possibility cannot be used for SNP partings for the obtained primer of design, lacks KASP at this stage and draws The possible designs scheme of thing.Meanwhile rs1048943, rs1051730, rs13181, rs1799793 of the present invention and Rs25487 is located at high GC content region, the difficulty that continuous GC is distributed with, further increases design of primers around it more.Application People finally just obtains primer of the present invention according to experience after paying a large amount of creative works, primer of the present invention Parting effect is good, is suitable for KASP technologies.

In some specific embodiments, the sequence label 1 is different with the nucleotide sequence of sequence label 2, described The nucleotide sequence of sequence label 1 such as SEQ ID NO：Shown in 21, the nucleotide sequence such as SEQ ID NO of the sequence label 2： Shown in 22.

The invention further relates to the kit for detecting foregoing SNP marker combination, the kit includes foregoing KASP Primer combines and other auxiliary detection reagents.

In some specific embodiments, other described auxiliary detection reagents include one or more of：Probe groups Conjunction, water, PCR buffer solutions, Mg²⁺, dNTPs and archaeal dna polymerase.

In some specific embodiments, the kit includes Mix, and the Mix includes the probe combinations, PCR Buffer solution, Mg²⁺, in dNTPs and archaeal dna polymerase at least two.

In some specific embodiments, the probe combinations include fluorescence probe 1~2 and quenching probes 1~2, its In, fluorescence probe 1 is made of sequence label 1 with the fluorophor 1 for being connected to its 5 ' end, and fluorescence probe 2 is by sequence label 2 and even The fluorophor 2 for being connected on its 5 ' end forms, and the fluorophor 1 and the fluorophor 2 send the fluorescence of different colours, described Quenching probes 1 are by the nucleotide sequence with 1 complementary pairing of sequence label and mark in its 3 ' fluorescent quenching group group held Into the quenching probes 2 are by the nucleotide sequence with 2 complementary pairing of sequence label and mark in its 3 ' fluorescent quenching held Group forms；

Preferably, the fluorophor 1 and the fluorophor 2 be selected from FAM, TET, JOE, HEX, CY3, TAMRA, ROX, Texas Red, LC RED640, LC RED705, the fluorescent quenching group be selected from Dabcyl, BHQ-1, QYS-7, BHQ-2 or ECLIPSE。

In some specific embodiments, the archaeal dna polymerase be selected from Taq, Bst, Vent, Phi29, Pfu, Tru, Tth, Tl1, Tac, Tne, Tma, Tih, Tf1, Pwo, Kod, Sac, Sso, Poc, Pab, Mth, Pho, ES4DNA polymerase, Klenow fragments.

Compared with prior art, beneficial effects of the present invention are：

(1) SNP marker of the present invention combination can system, comprehensively reflect lung cancer susceptibility, be particularly suitable in China Crowd uses, and contributes to early detection lung cancer population, so as to targetedly better people's living environment and habits and customs, lowers lung cancer Incidence.

(2) primer combination of the present invention is that for KASP detection methods, specially exploitation is used to detect foregoing SNP applicant The primer of mark.Wherein be used for detect each SNP site primer include two with sequence label sense primers and without The anti-sense primer of sequence label, it coordinates with the fluorescence probe and quenching probes for being connected to fluorophor and quenching group and makes With, can be by the polymorphism of the above-mentioned SNP marker of KASP technology for detection, pair with accuracy rate height, flexibility by force, without costliness It is color fluorescence labeling probe, of less demanding to sample and the advantages of can realize the detection of big flux.

(3) applicant overcomes the prior art to lack feasible KASP design of primers schemes, and SNP of the present invention is located at High GC content region, around it that continuous GC is distributed with, design of primers difficulty is big to wait technical barrier, according to experience, pays more The final primer that can be used for KASP technologies, parting effect good that obtains combines after going out a large amount of creative works.

Brief description of the drawings

, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution of the prior art Embodiment or attached drawing needed to be used in the description of the prior art are briefly described, it should be apparent that, in describing below Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor Put, other attached drawings can also be obtained according to these attached drawings.

Fig. 1 is the result that rs1048943 Genotypings are carried out using primer 1~3 described in embodiment 2；

Fig. 2 is the result that rs1051730 Genotypings are carried out using primer 4~6 described in embodiment 2；

Fig. 3 is the result that rs13181 Genotypings are carried out using primer 7~9 described in embodiment 2；

Fig. 4 is the result that rs1799793 Genotypings are carried out using primer 10~12 described in embodiment 2；

Fig. 5 is the result that rs25487 Genotypings are carried out using primer 13~15 described in embodiment 2；

Fig. 6 is the result that rs1048943 Genotypings are carried out using primer described in comparative example 1 (SEQ ID 25~27)；

Fig. 7 is the result that rs1051730 Genotypings are carried out using primer described in comparative example 1 (SEQ ID 28~30)；

Fig. 8 is the result that rs25487 Genotypings are carried out using primer described in comparative example 1 (SEQ ID 31~33).

Embodiment

Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific Condition person, the condition suggested according to normal condition or manufacturer carry out.Reagents or instruments used without specified manufacturer, is The conventional products obtained can be bought by city.

1 sequence of embodiment is searched

NCBI is logged in, inquires about the corresponding sequence of SNP site, the particular sequence such as institute of table 1 in GenBank according to SNP site Show.

The corresponding allelic variation sequence of 1 SNP site of table

2 design of primers of embodiment and synthesis

For SNP site described in embodiment 1, design specialized in the primer combination of KASP technologies and probe combinations, wherein, institute Stating primer includes being exclusively used in the primer combination of foregoing 5 SNP sites detection, and the probe combinations include fluorescence probe 1~2 and quench Go out probe 1~2, and wherein the nucleotide sequence of fluorescence probe 1,2 is identical with sequence label 1,2 respectively, the core of quenching probes 1~2 Nucleotide sequence is respectively the nucleotide sequence of sequence label 1,2 reverse complementals (particular sequence is referring to table 2).

Foregoing primer and probe is synthesized, and flag F AM, 5 ' end mark HEX of fluorescence probe 2 are held the 5 ' of fluorescence probe 1, With 3 ' end mark BHQ of quenching probes 1,2.

KASP primer specials, label and the probe sequence that 2 present invention of table develops

The foundation of embodiment 3KASP detection methods

Using the individual DNA to be detected of extraction as template, primer special, the probe of the KASP marks developed with embodiment 2 Carry out PCR amplification and detection.Specific method is as follows：

1) Primer Mix are prepared according to ratio shown in table 3,4 degree of preservations of refrigerator is placed on after preparing.

The ratio of each component in 3 Primer Mix of table

Primer	Volume
		Sense primer 1 (100mM)	12μL
Sense primer 2 (100mM)	12μL
		Anti-sense primer (100mM)	30μL
H2O	46μL
		total	100μL

2) people's DNA profiling to be checked (sample is from buccal swab or blood) (20-30ng/ μ L) is added in 384 orifice plates, Per 2.5 μ L of hole.

3) (calculated according to proportional arrangement Assay Mix shown in table 4 according to sample number), wherein, the Master Mix Including fluorescence probe 1~2, quenching probes 1~2, DNA Polymerase, dNTP, buffer solution and Mg²⁺Deng progress PCR amplification institute The component needed.

The ratio of each component in 4 Assay Mix of table

	1	100
			Master Mix(2×)	2.5μL	250μL
Primer mix	0.07μL	7μL
			Total	2.57μL	257μL

4) prepared Assay Mix are dispensed into 384 orifice plates for having added template, 2.5 μ L/ holes.Remarks：Experiment The blank control for not adding template DNA in reaction system is set at the same time, and each PCR plate sets a blank control.

5) 384 orifice plate special sealing membranes are covered, and are compressed.

6) centrifuge, 4000rpm 4min, makes liquid all sink to tube bottom.

7) it is put into PCR instrument, reaction condition carries out Touch Down PCR as shown in table 5.

The specific reaction condition of 5 Touch Down PCR of table

8) after the completion of PCR, 384 orifice plates are centrifuged, 4000rpm, 4min.Referring next to《ABI7900HT Fast Realtime PCR system experimental implementation codes》Data scanning is carried out, obtains the genotype of each sample.According to analysis result According to the specific genotype that each site is identified below：The genotype being aggregated in close to the sample of X-coordinate axle is connection FAM fluorescence marks The allelotype of sequence is signed, is aggregated in the equipotential for connection HEX fluorescence labels sequences close to the sample genotype in Y-coordinate axle Genotype, the sample genotype positioned at middle diagonal positions are the heterozygous of two kinds of allele.Specific testing result such as Fig. 1 Shown in~5.According to result shown in Fig. 1~5, testing result agglomerating well can be gathered in X-axis, Y-axis or middle right At diagonal position, parting effect is good.

Comparative example 1

Carry out KASP detections with reference to embodiment 3 the method, differ only in, the method detection rs1048943, During the polymorphism of rs1051730 and rs25487, the primer used is as shown in table 6.Specific testing result as can be seen from figures 6 to 8, root Understand that testing result random distribution in X, Y direction, software can not be carried out according to testing result according to result shown in Fig. 6~8 Parting, parting effect are poor.

6 KASP primer special sequences of table

Finally it should be noted that：The above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent Pipe is described in detail the present invention with reference to foregoing embodiments, but it will be understood by those of ordinary skill in the art that：Its It can still modify to the technical solution described in foregoing embodiments, either to which part or all technical characteristic Carry out equivalent substitution；And these modifications or replacement, the essence of appropriate technical solution is departed from various embodiments of the present invention skill The scope of art scheme.

SEQUENCE LISTING

<110>Sinogenomax Co., Ltd.

<120>SNP marker for detecting lung cancer susceptibility combines, primer combines and kit

<160> 33

<170> PatentIn version 3.3

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Claims (10)

1. the SNP marker combination for detecting lung cancer susceptibility, it is characterised in that the SNP marker combination includes Rs1048943, rs1051730, rs13181, rs1799793 and rs25487.

2. the reagent for the SNP marker combination of test right requirement 1 is being prepared for detecting in the reagent of lung cancer susceptibility Application.

3. application according to claim 2, it is characterised in that the reagent is used for ApoE gene, sequencing One in method, Taqman sonde methods, Single strand conformation polymorphism, denaturing gradient gel electrophoresis or high-resolution solubility curve method Kind is a variety of；

Preferably, the reagent is used for ApoE gene；

It is highly preferred that the ApoE gene is competitive ApoE gene.

4. the KASP (Kompetitive Allele Specific PCR) for the SNP marker combination of test right requirement 1 Primer combines, it is characterised in that the primer combination includes：

The primer 1~3 of rs1048943 is detected, the primer 1 is from 5 ' ends to 3 ' ends by sequence label 1 and such as SEQ ID NO：6 institutes Show that nucleotide sequence forms, the primer 2 is from 5 ' ends to 3 ' ends by sequence label 2 and such as SEQ ID NO：Nucleotides sequence shown in 7 Row composition, the primer 3 is from 5 ' ends to 3 ' ends by SEQ ID NO：Nucleotide sequence shown in 8 forms；

The primer 4~6 of rs1051730 is detected, the primer 4 is from 5 ' ends to 3 ' ends by sequence label 1 and such as SEQ ID NO：9 institutes Show that nucleotide sequence forms, the primer 5 is from 5 ' ends to 3 ' ends by sequence label 2 and such as SEQ ID NO：Nucleotides sequence shown in 10 Row composition, the primer 6 is from 5 ' ends to 3 ' ends by SEQ ID NO：Nucleotide sequence shown in 11 forms；

The primer 7~9 of rs13181 is detected, the primer 7 is from 5 ' ends to 3 ' ends by sequence label 1 and such as SEQ ID NO：12 institutes Show that nucleotide sequence forms, the primer 8 is from 5 ' ends to 3 ' ends by sequence label 2 and such as SEQ ID NO：Nucleotides sequence shown in 13 Row composition, the primer 9 is from 5 ' ends to 3 ' ends by SEQ ID NO：Nucleotide sequence shown in 14 forms；

The primer 10~12 of rs1799793 is detected, the primer 10 is from 5 ' ends to 3 ' ends by sequence label 1 and such as SEQ ID NO： Nucleotide sequence shown in 15 forms, and the primer 11 is from 5 ' ends to 3 ' ends by sequence label 2 and such as SEQ ID NO：Core shown in 16 Nucleotide sequence forms, and the primer 12 is from 5 ' ends to 3 ' ends by SEQ ID NO：Nucleotide sequence shown in 17 forms；

With the primer 13~15 of detection rs25487, the primer 13 is from 5 ' ends to 3 ' ends by sequence label 1 and such as SEQ ID NO： Nucleotide sequence shown in 18 forms, and the primer 14 is from 5 ' ends to 3 ' ends by sequence label 2 and such as SEQ ID NO：Core shown in 19 Nucleotide sequence forms, and the primer 15 is from 5 ' ends to 3 ' ends by SEQ ID NO：Nucleotide sequence shown in 20 forms.

5. KASP primers combination according to claim 4, it is characterised in that the core of the sequence label 1 and sequence label 2 Nucleotide sequence is different, the nucleotide sequence such as SEQ ID NO of the sequence label 1：Shown in 21, the nucleosides of the sequence label 2 Acid sequence such as SEQ ID NO：Shown in 22.

6. the kit for the SNP marker combination of test right requirement 1, it is characterised in that the kit includes right It is required that the combination of 4~5 any one of them KASP primers and other auxiliary detection reagents.

7. kit according to claim 6, it is characterised in that other described auxiliary detection reagents include it is following a kind of or It is a variety of：Probe combinations, water, PCR buffer solutions, Mg²⁺, dNTPs and archaeal dna polymerase.

8. kit according to claim 7, it is characterised in that the kit includes Mix, and the Mix includes probe Combination, PCR buffer solutions, Mg²⁺, in dNTPs and archaeal dna polymerase at least two.

9. kit according to claim 7, it is characterised in that the probe combinations include fluorescence probe 1~2 and are quenched Probe 1~2, wherein, fluorescence probe 1 is made of sequence label 1 with the fluorophor 1 for being connected to its 5 ' end, and fluorescence probe 2 is by marking Label sequence 2 is formed with the fluorophor 2 for being connected to its 5 ' end, and the fluorophor 1 and the fluorophor 2 send different colours Fluorescence, the quenching probes 1 by the nucleotide sequence with 1 complementary pairing of sequence label and mark its 3 ' end fluorescence Quenching group forms, and the quenching probes 2 are by the nucleotide sequence with 2 complementary pairing of sequence label and mark at its 3 ' end Fluorescent quenching group composition；

Preferably, the fluorophor 1 and the fluorophor 2 be selected from FAM, TET, JOE, HEX, CY3, TAMRA, ROX, Texas Red, LC RED640, LC RED705, the fluorescent quenching group be selected from Dabcyl, BHQ-1, QYS-7, BHQ-2 or ECLIPSE。

10. kit according to claim 7, it is characterised in that the archaeal dna polymerase be selected from Taq, Bst, Vent, Phi29、Pfu、Tru、Tth、Tl1、Tac、Tne、Tma、Tih、Tf1、Pwo、Kod、Sac、Sso、Poc、Pab、Mth、Pho、 ES4DNA polymerases, Klenow fragments.