CN107513574A - Detect the primer set of rs11190870 polymorphism and its application in human genome - Google Patents

Abstract

The invention discloses the primer set of rs11190870 polymorphism and its application in detection human genome.The primer set of rs11190870 polymorphism is made up of four primers that title is respectively AIS_Fin, AIS_Rin2, AIS_F and AIS_R in the detection human genome, the AIS_Fin is the single stranded DNA shown in SEQ ID No.1, the AIS_Rin2 is the single stranded DNA shown in SEQ ID No.2, the AIS_F is the single stranded DNA shown in SEQ ID No.3, and the AIS_R is the single stranded DNA shown in SEQ ID No.4.The present invention shortens detection cycle, reduces testing cost compared with Sanger is detected, available for the examination and prevention for finding AIS people at highest risk and adolescent idiopathic scoliosis.

Description

Detect the primer set of rs11190870 polymorphism and its application in human genome

Technical field

The present invention relates in biological technical field detect human genome in rs11190870 polymorphism primer set and It is applied.

Background technology

Scoliosis, also known as scoliosis, it is that one kind is mainly in teen-age spinal lesion, has a strong impact on teen-age body The heart develops in a healthy way.Scoliosis is a kind of disease of long-run development, once occur after, can along with it is teen-age grow by Walk development, deteriorate, while also result in the increasing for the treatment of difficulty, therapeutic effect variation.Therefore, the early detection of scoliosis, dry It is pre- especially important.At present, for the examination of teenagers scoliosis, the most frequently used means are to take the mode of field screening, right The hair patient for having been detected by scoliosis is intervened, treated.But due to early stage, scoliosis degree occurs in scoliosis It is small, it is not easy to it is detected, so conventional examination can omit the morbidity patient of some early stages, meanwhile, using the sieve of routine Checking method consumes a large amount of manpower and materials, it is necessary to repeat at periodic or other desired inspection, and therefore, use is more economical, effective method is to the people that falls ill Group screen most important.

In teenagers scoliosis morbidity crowd, incidence of disease highest is adolescent idiopathic scoliosis (adolescent idiopathic scoliosis, AIS), account for the 80% of morbidity total number of persons.Hair on AIS in recent years In sick Mechanism Study, inherent cause gradually obtains the accreditation of most researchers, the SNP site related to AIS morbidities also progressively by It was found that wherein, can most obtain researcher's accreditation is a SNP site near LBX1 genes：rs11190870. Hua Jiang et al. are right in Chinese Han Population AIS research, incorporating 949 AIS patients and 976 health altogether at one According to crowd, result of study is shown, rs11190870 C ＞ T variations and significantly correlated (P=1.89 × 10 of AIS morbidities^-9, OR= 1.51)(Jiang H,Qiu XS,Dai J et al.Association of rs11190870near LBX1 with adolescent idiopathic scoliosis susceptibility in a Han Chinese population.Eur Spine J(2013)22:282–286)；In addition, Yaqin Cao et al. incorporate it is multinomial on AIS's Research has carried out meta analyses, the results showed that, rs11190870 T genotype can increase population of adolescent, particularly women people Onset risk (Cao YQ, the Min JK et al.Associations of LBX1 gene and adolescent of group idiopathic scoliosis susceptibility:a meta-analysis based on 34, 626subjects.Cao et al.BMC Musculoskeletal Disorders(2016)17:309).So pass through base Because of the mode of detection, lock adolescent idiopathic scoliosis group of people at high risk and carry out emphasis examination, and formulate targetedly Intervening measure, while screening difficulty is reduced, the effect of examination can also be improved.

In being detected currently for human body SNP Genotyping, detected for single or small number of SNP, it is conventional Detection technique be mainly Mass Spectrometer Method, implement fluorescent quantitative PCR technique, Sanger sequencing etc., wherein, Sanger PCR sequencing PCRs are the most It is common.It is higher to laboratory hardware requirement due to carrying out Sanger sequencings at present, so most laboratory is produced with sending PCR outside at present Thing is carried out based on the mode of Sanger sequencings, by sequencing company on behalf of detection.This subject matter sent sequencing mode outside and brought Poor in timeliness is exactly detected, detection cycle is generally longer.Meanwhile although at present single sample carry out Sanger sequencings price compared with It is low, but for the larger laboratory of sample, testing cost is still a burden.In addition, Mass Spectrometer Method, implementation quantitative fluorescent PCR Technology also has higher requirement to laboratory hardware device.So a kind of quick, economic SNP site detection method of exploitation becomes Obtain very necessary.

2001, principles of the Ye et al. based on ARMS-PCR, one kind is introduced in laboratory quick detection target site sequence The method of row, i.e. four primers variation Retardation of amplification system (Tetra-primer ARMS PCR), inside and outside target site design Totally 4 primers, wherein 2 outer primers (F, R) are respectively positioned at target site upstream, downstream；Other 2 inner primer sequences direction phase Instead, and the end of sequence 3 ' is located at target SNP site, corresponds to two kinds of genotype (Ye S, Dhillon S, Ke X, et respectively al.An efficient procedure for genotyping single nucleotide polymorphisms[J] .Nucleic Acids Res,2001,29(17):e88).2 outer primers, 2 inner primers are mixed into performing PCR to expand, two kinds PCR primer fragment length corresponding to genotype is different, and target site is can determine that by the band distribution of electrophoresis detection PCR primer Genotype (wild homozygosis, heterozygosis, homozygous mutation).In ARMS-PCR technologies, primer is the pass for determining the testing result degree of accuracy Key factor.

The content of the invention

The technical problems to be solved by the invention are how to detect rs11190870 in human genome exactly polymorphic Property (i.e. allele).

In order to solve the above-mentioned technical problem, the invention provides the polymorphism of rs11190870 in detection human genome (i.e. Allele) primer set.

The primer set of rs11190870 polymorphism is respectively by title in detection human genome provided by the present invention AIS_Fin, AIS_Rin2, AIS_F and AIS_R four primers composition, the AIS_Fin is the list shown in SEQ ID No.1 Chain DNA, the AIS_Rin2 are the single stranded DNAs shown in SEQ ID No.2, and the AIS_F is single-stranded shown in SEQ ID No.3 DNA, the AIS_R are the single stranded DNAs shown in SEQ ID No.4.

In above-mentioned primer set, the proportioning of four primers can be 2nmol AIS_Fin：2nmol AIS_Rin2： 1nmol AIS_F：1nmol AIS_R.

The reagent of rs11190870 polymorphism (i.e. allele) in detection human genome containing above-mentioned primer set Or kit falls within protection scope of the present invention.

The reagent of rs11190870 polymorphism or kit may also include in above-mentioned detection human genome expands into performing PCR Required other reagents.

Above-mentioned primer set is preparing the reagent for the polymorphism (i.e. allele) for detecting rs11190870 in human genome Or the application in kit falls within protection scope of the present invention.

Above-mentioned primer set or mentioned reagent or kit are preparing examination adolescent idiopathic scoliosis excessive risk people Application in group's product (reagent or kit) falls within protection scope of the present invention.

Present invention also offers the method using rs11190870 genotype in above-mentioned primer set detection human genome.

The method of rs11190870 genotype in detection human genome provided by the present invention, including the base with people to be measured Because group DNA is template, enters performing PCR using above-mentioned primer set and expand to obtain PCR primer, detect the size in the PCR primer More than 100bp and less than the composition of the DNA fragmentation between 500bp, the genotype of rs11190870 in human genome is determined；Will Size in the PCR primer is being named as L100-500 section products more than 100bp and less than the DNA fragmentation between 500bp, If the L100-500 sections product is by L189 and L411, the two DNA fragmentations form, in the genome of the people to be detected Rs11190870 genotype is TT, if the L100-500 sections product is by L273 and the L411 the two DNA fragmentations Composition, rs11190870 genotype is CC in the genome of the people to be detected, if the L100-500 sections product by The L189, the L273 and the L411 these three DNA fragmentations composition, rs11190870 in the genome of the people to be detected Genotype be TC；The L189 is greater than the DNA fragmentation that 100bp is less than 250bp, and it is small that the L273 is greater than 250bp In a 500bp DNA fragmentation, the L411 is greater than the DNA fragmentation that 250bp is less than 500bp.

In the above method, a L189 concretely 189bp DNA fragmentation, the L273 concretely 273bp One DNA fragmentation, a L411 concretely 411bp DNA fragmentation.

In the above method, the primer annealing condition that the PCR amplifications use is 57 DEG C of annealing 30s.

In the above method, the PCR temperature programmings that are used in PCR amplification：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 57 DEG C of annealing 30s, 72 DEG C of extension 30s, 30-35 circulates；72 DEG C of extension 2min.

The final purpose of the above method can be medical diagnosis on disease purpose, disease prognosis purpose and/or disease treatment purpose, on The final purpose for stating method can also be non-medical diagnosis on disease purpose, non-disease prognosis purpose and non-disease therapeutic purposes；Above-mentioned side The direct purpose of method can obtain the intermediate result of medical diagnosis on disease result, disease prognosis result and/or disease treatment result Information, the direct purpose of the above method can treat mesh with right and wrong medical diagnosis on disease purpose, non-disease prognosis purpose and/or non-disease 's.

Herein, rs11190870 is the SNP site of a two equipotential polymorphisms on human chromosome, and the variation is conversion (C/T, being then G/A on its complementary strand).The rs11190870 genotype is CC, TC or TT.The CC is rs11190870 Site is the homozygous of C, and the TT is that rs11190870 sites are the homozygous of T, the TC be rs11190870 sites for C and T heterozygous.Rs11190870 polymorphism (i.e. allele) or genotype are concretely examined in the detection human genome Survey rs11190870 nucleotides species.

Four primer variations are carried out using the primer set of rs11190870 polymorphism in the detection human genome of the present invention Retardation of amplification, the rs11190870 of people to be measured genotype can be distinguished exactly according to amplified production.Made a variation based on four primers Retardation of amplification system, using the primer set of rs11190870 polymorphism in the detection human genome of the present invention, one can be established Method of the kind for AIS onset risk SNP sites rs11190870 rapid gene parting.The rapid gene of the rs11190870 The method of parting includes buccal swab DNA extractions, the amplification of four primers variation Retardation of amplification system, the Ago-Gel of PCR primer Electrophoresis detection, according to electrophoresis pattern, you can directly to judge to be detected the genotype of sample.The rapid gene of the rs11190870 The method of parting can complete whole testing processes in 8 hours, compared to traditional Sanger sequence measurements, substantially reduce inspection Survey the cycle.Simultaneously as the technology is only detected by the agarose gel electrophoresis of PCR primer, you can to judge the gene of sample Type, therefore more traditional Sanger is detected, and greatly reduces testing cost.The present invention can be used for find AIS people at highest risk and The prevention of adolescent idiopathic scoliosis.

Brief description of the drawings

Fig. 1 is the Sanger sequencing results of TT genotype sample, CC genotype sample and TC genotype samples.

Fig. 2 is that five kinds of primer sets enter the PCR primer electrophoresis result that performing PCR obtains to three kinds of genotype samples.

In Fig. 2, swimming lane M, which is D2000 DNA Marker, c1, to be respectively primer set A, primer set B, complete draws to c4 Thing C and primer set D enters the PCR primer electrophoresis result that performing PCR obtains to TT genotype samples, and c5 to c8 is respectively primer set A, primer set B, primer set C and primer set D enter the PCR primer electrophoresis result that performing PCR obtains to CC genotype samples；c9 It is respectively that primer set A, primer set B, primer set C and primer set D enter performing PCR to TC genotype samples and obtained to c12 PCR primer electrophoresis result；C13 to c16 is respectively primer set A, primer set B, primer set C and primer set D to water Enter the PCR primer electrophoresis result that performing PCR obtains.

Embodiment

The present invention is further described in detail with reference to embodiment, the embodiment provided is only for explaining The bright present invention, the scope being not intended to be limiting of the invention.Experimental method in following embodiments, unless otherwise specified, it is Conventional method.Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.

Rs11190870 polymorphism (the Genotyping inspection in rs11190870 sites in embodiment 1, detection human genome Survey)

1st, the design of four primers variation Retardation of amplification system primer

Sequence corresponding to rs11190870, each 500bp of the SNP site upstream and downstream, total length are downloaded from ncbi database 1001bp.Rs11190870 genotype includes tri- kinds of TT, TC, CC, and the design website provided by Ye et al. carries out design of primers, Website is：http://primer1.soton.ac.uk/primer1.html.This is named using the primer of Software for Design It is made up of for primer set A, primer set A following A IS_Fin, AIS_Rin, AIS_F and AIS_R：

1. inner primer 1, T allele corresponds to primer, entitled AIS_Fin, and sequence is：

5’-AGGAATTATCAACTAGAATTTGATTAAGAT-3’(SEQ ID No.1)。

2. inner primer 2, C allele corresponds to primer, entitled AIS_Rin, and sequence is：

5’-AGCTGTTTGCCTGCGATTTTCG-3’。

3. outer primer 1, Forward primers, entitled AIS_F, sequence are：

5’-ATCATAAAACCGGACAGGCAATT-3’(SEQ ID No.3)。

4. outer primer 2, Reverse primers, entitled AIS_R, sequence are：

5’-CGTTTCTGCAGAGCCTCTTAATACTT-3’(SEQ ID No.4)。

Wherein, the T allele specifics product length of the estimated amplifications of AIS_Fin and AIS_R be 189bp, AIS_Rin with The C allele specifics product length of the estimated amplifications of AIS_F is including for the estimated amplification of 273bp, AIS_F and AIS_R Rs11190870 specific product length is 411bp.

Primer set A is tested in experiment, it is found that primer set A specific effect is poor, for T etc. Easily there is false positive in the detection of position gene, therefore the present inventor is improved to primer set A, obtains primer set B, complete draws Thing C and primer set D.Primer set A, primer set B, primer set C and primer set D form by four primers, they Differ only in inner primer 2 (C allele corresponds to primer), other three primers (AIS_Fin, AIS_F, AIS_R) are complete It is identical.Primer set B is made up of AIS_Fin, AIS_F, AIS_R and AIS_Rin2 (inner primer 2), and primer set C is by AIS_ Fin, AIS_F, AIS_R and AIS_Rin3 (inner primer 2) are formed, and primer set D is by AIS_Fin, AIS_F, AIS_R and AIS_ Rin4 (inner primer 2) is formed.

Wherein, it is as follows to correspond to primer AIS_Rin2, AIS_Rin3 and AIS_Rin4 information for C allele：

AIS_Rin2, the former end of AIS_Rin primers 3 ' are changed to by the 3rd bit mismatch：The strong mispairing of second G-A, sequence are：

5’-AGCTGTTTGCCTGCGATTTGAG-3’(SEQ ID No.2)；

AIS-Rin3, former AIS_Rin primers 3 ' are kept to hold the 3rd bit mismatch constant, second introduces the weak mispairing of G-T, sequence For：5’-AGCTGTTTGCCTGCGATTTTTG-3’；

2 bases are reduced by AIS-Rin4, the former end of AIS_Rin primers 5 ', and 3 ' ends are changed to by the 3rd bit mismatch：Second G-A Strong mispairing, sequence are：5’-CTGTTTGCCTGCGATTTGAG-3’.

In primer set B, the T allele specifics product length of AIS_Fin and the estimated amplifications of AIS_R is 189bp, AIS_ The C allele specifics product length of the estimated amplifications of Rin2 and AIS_F is including for the estimated amplification of 273bp, AIS_F and AIS_R Rs11190870 specific product length is 411bp.

In primer set C, the T allele specifics product length of AIS_Fin and the estimated amplifications of AIS_R is 189bp, AIS_ The C allele specifics product length of the estimated amplifications of Rin3 and AIS_F is including for the estimated amplification of 273bp, AIS_F and AIS_R Rs11190870 specific product length is 411bp.

In primer set D, the T allele specifics product length of AIS_Fin and the estimated amplifications of AIS_R is 189bp, AIS_ The C allele specifics product length of the estimated amplifications of Rin4 and AIS_F is including for the estimated amplification of 271bp, AIS_F and AIS_R Rs11190870 specific product length is 411bp.

2nd, the preparation of TT genotype sample, CC genotype sample and TC genotype samples

Human Oral Cavity swab sample is gathered, each 2 swabs of sample collection, carries out human gene group DNA's extraction, is used Qubit2.0 detects DNA concentration.

Enter performing PCR amplification to human gene group DNA using AIS_F and AIS_R, after electrophoresis detection is qualified, carry out Sanger surveys Sequence, according to sequencing result, using the human gene group DNA of TT genotype (rs11190870 sites are the homozygous of T) as TT genes Pattern sheet, using the human gene group DNA of CC genotype (rs11190870 sites be C homozygous) as CC genotype sample, general The human gene group DNA of TC genotype (rs11190870 sites are C and T heterozygous) is as TC genotype sample (Fig. 1).

3rd, the optimization of PCR reaction conditions

After primer synthesis, grope the annealing temperature of PCR system, in four primers of primer set, select minimum Tm It is worth to refer to, 2 DEG C are gradient, and 3 gradients of design are tested, the use that selector bar band is clear, annealing temperature is higher.

Reacted for PCR, reagent uses 2 × Taq MasterMix (the Dye) (Cat of health for century：CW0682M), DNA Sample size is 20ng, and reaction system is 25 μ L, and inside and outside primer final concentration is respectively 0.4 μM, 0.2 μM, PCR temperature programmings Such as table 1：

Table 1, PCR temperature programmings

For PCR primer, detected using agarose gel electrophoresis, deposition condition is：1.5% Ago-Gel, 1% TE electrophoresis liquids, 100V, 50 minutes；PCR primer electrophoresis applied sample amount is 5 μ L.

Tiangeng D2000DNA Marker (Cat are used during electrophoresis detection：MD114 standard items) are used as, its band is from big to small It is followed successively by：2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp.

Tm values temperature is minimum for AIS_Fin in primer set A, designs three annealing temperatures with reference to its Tm value, is respectively 53℃、55℃、57℃.Performing PCR amplification is entered to the TT genotype sample, CC genotype sample and TC genotype samples of step 2, it is right PCR primer carries out electrophoresis detection, and electrophoresis detection result shows to remain to target stripe at amplification when temperature is 57 DEG C, therefore selects 57 DEG C be used as final annealing temperature.

4th, primer set A, primer set B, primer set C and primer set D detection TT genotype sample, CC gene patterns Sheet and TC genotype samples

Made respectively using TT genotype sample, CC genotype sample and the TC genotype sample of step 2 as template, while with water For control, it is utilized respectively primer set A, primer set B, primer set C and primer set D and enters performing PCR amplification.

Pcr amplification reaction system is as follows：25 μ L reaction systems：The μ of DNA profiling 20ng, 2 × Taq MasterMix 12.5 L, 0.4 μM of 1 final concentration of inner primer, 0.4 μM of 2 final concentration of inner primer, 0.2 μM of 1 final concentration of outer primer, the μ of 2 final concentration of outer primer 0.2 M, nuclease-free water complements to 25 μ l.

PCR temperature programmings are such as table 2：

Table 2, PCR temperature programmings

Detection PCR primer is carried out using agarose gel electrophoresis, deposition condition is：1.5% Ago-Gel, 1%TE Electrophoresis liquid, 100V, 40 minutes；PCR primer electrophoresis applied sample amount is 5 μ L.

Tiangeng D2000DNA Marker (Cat are used during electrophoresis detection：MD114 standard items) are used as, its band is from big to small It is followed successively by：2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp.

Electrophoresis result is as shown in Fig. 2 from glue it can be seen from the figure that, the primer set A of design of primers website design detects TT During genotype sample, non-specific amplification be present in sections of the 100bp extremely less than 500bp, be easily mistaken for TC (see in Fig. 2 Swimming lane c1)；For TT genotype samples, 411bp bands are weaker in primer set C PCR primer, influence result interpretation (see Fig. 2 Middle swimming lane c3)；For CC genotype samples, the 273bp bands and 411bp bands in primer set C PCR primer are weaker, Influence result interpretation (see swimming lane c7 in Fig. 2)；For TC genotype samples, primer set C PCR primer and primer set D's PCR primer band is weaker, and particularly 273bp band is very weak, influences result interpretation (see swimming lane c11 and c12 in Fig. 2).Into Primer B is covered for TT genotype sample, CC genotype sample and TC genotype samples, and its PCR primer is (see swimming lane c2, c6 in Fig. 2 And c10) in be more than 100bp very clear to specific band in the section less than 500bp and without non-specific band, though Right primer set B PCR primer also has a non-specific amplification in about 500bp, but due to more than 100bp to being less than There is no non-specific amplification in 500bp section, do not influence the Genotyping detection in rs11190870 sites.It can be seen that above-mentioned four In kind primer set, primer set B is adapted to the Genotyping detection in rs11190870 sites the most.

In this technology research, complete the drawing of initially use four primers variation Retardation of amplification system design of primers website designs Thing A amplification TT genotype sample, CC genotype sample and TC genotype samples, there is non-specific amplification in discovery, easily by TT Genotype flase drop is TC.Therefore, this research is directed to the primer that non-specific amplification be present and is optimized, improves, and is used in combination Three kinds of genotype samples (TT, CC, TC) of Sanger checkings test the Detection results of four kinds of primer sets, finally determine into The Genotyping detection that primer B is adapted to rs11190870 sites is covered, available for examination AIS people at highest risk.

Primer set B is by following 1) -4) four primers form：

1) inner primer 1, T allele correspond to primer, entitled AIS_Fin, and sequence is：

5’-AGGAATTATCAACTAGAATTTGATTAAGAT-3’(SEQ ID No.1)。

2) inner primer 2, C allele correspond to primer, entitled AIS_Rin2, and sequence is：

5’-AGCTGTTTGCCTGCGATTTGAG-3’(SEQ ID No.2)。

3) outer primer 1, Forward primers, entitled AIS_F, sequence are：

5’-ATCATAAAACCGGACAGGCAATT-3’(SEQ ID No.3)。

4) outer primer 2, Reverse primers, entitled AIS_R, sequence are：

5’-CGTTTCTGCAGAGCCTCTTAATACTT-3’(SEQ ID No.4)。

In primer set B, the proportioning of four primers is 2nmol AIS_Fin：2nmol AIS_Rin2：1nmol AIS_F： 1nmol AIS_R。

Sequence table

<120>Detect the primer set of rs11190870 polymorphism and its application in human genome

<130> GNCFH171770

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 30

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 1

aggaattatc aactagaatt tgattaagat 30

<210> 2

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 2

agctgtttgc ctgcgatttg ag 22

<210> 3

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 3

atcataaaac cggacaggca att 23

<210> 4

<211> 26

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 4

cgtttctgca gagcctctta atactt 26

Claims (10)

1. detect the primer set of rs11190870 polymorphism in human genome, it is characterised in that：The primer set is by name Claim respectively AIS_Fin, AIS_Rin2, AIS_F and AIS_R four primers composition, the AIS_Fin is SEQ ID No.1 Shown single stranded DNA, the AIS_Rin2 are the single stranded DNAs shown in SEQ ID No.2, and the AIS_F is SEQ ID No.3 institutes The single stranded DNA shown, the AIS_R are the single stranded DNAs shown in SEQ ID No.4.

2. primer set according to claim 1, it is characterised in that：The proportioning of four primers described in the primer set For 2nmol AIS_Fin：2nmol AIS_Rin2：1nmol AIS_F：1nmol AIS_R.

3. detect the reagent or kit of rs11190870 polymorphism in human genome, it is characterised in that：The reagent or examination Agent box includes the primer set described in claim 1 or 2.

4. the primer set described in claim 1 or 2 is preparing the reagent for the polymorphism for detecting rs11190870 in human genome Or the application in kit.

5. the answering in examination adolescent idiopathic scoliosis patient product is prepared of the primer set described in claim 1 or 2 With.

6. the answering in examination adolescent idiopathic scoliosis patient product is prepared of the reagent or kit described in claim 3 With.

7. the method for rs11190870 genotype in human genome is detected, including using the genomic DNA of people to be measured as template, profit Enter performing PCR with the primer set described in claim 1 or 2 to expand to obtain PCR primer, the size detected in the PCR primer exists More than 100bp and less than the composition of the DNA fragmentation between 500bp, the genotype of rs11190870 in human genome is determined；By institute The size stated in PCR primer is being named as L100-500 section products more than 100bp and less than the DNA fragmentation between 500bp, such as By L189 and L411, the two DNA fragmentations form L100-500 sections product described in fruit, in the genome of the people to be detected Rs11190870 genotype is TT, if the L100-500 sections product is by L273 and the L411 the two DNA fragmentations Composition, rs11190870 genotype is CC in the genome of the people to be detected, if the L100-500 sections product by The L189, the L273 and the L411 these three DNA fragmentations composition, rs11190870 in the genome of the people to be detected Genotype be TC；The L189 is greater than the DNA fragmentation that 100bp is less than 250bp, and it is small that the L273 is greater than 250bp In a 500bp DNA fragmentation, the L411 is greater than the DNA fragmentation that 250bp is less than 500bp.

8. according to the method for claim 7, it is characterised in that：The L189 is a 189bp DNA fragmentation, described L273 is a 273bp DNA fragmentation, and the L411 is a 411bp DNA fragmentation.

9. according to the method for claim 8, it is characterised in that：The primer annealing condition that the PCR amplifications use is 57 DEG C Anneal 30s.

10. method according to claim 8 or claim 9, it is characterised in that：The PCR reaction temperature journeys used in the PCR amplifications Sequence：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 57 DEG C of annealing 30s, 72 DEG C of extension 30s, 30-35 circulates；72 DEG C of extensions 2min。