CN105861731A - Detection kit for genes related to sport advantages of Chinese people and application thereof - Google Patents

Abstract

The invention discloses a detection kit for genes related to sport advantages of Chinese people. The kit comprises an insert or delete (I/D) polymorphism detection primer of 16th intron of the ACE gene and an R577X polymorphism detection primer of the ACTN3 gene. The kit is specially used for detecting the sport advantage genes of Chinese people and can serve as a novel method for selecting or matching a sport or sport event (such as a competitive event, velocity acceleration, strength-typed sport event and a endurance-typed sport event) for an individual and evaluating and predicating sport performance and optimizing and designing training plans, and the method includes evaluation of multiple genes or gene types. The kit can also be used for detecting twelve genes related to sport advantages and potentials of infants, children and teenagers, predicating sport gift of the infants, children and teenagers and providing reasonable advices for sport events on which the infants, children and teenagers work in the future.

Description

The detection kit of Chinese's motion Dominant Facies correlation gene and application thereof

Technical field

The present invention relates to detection kit and the application thereof of a kind of motion Dominant Facies correlation gene, be particularly suitable for Chinese The detection kit of crowd, belongs to technical field of biological.

Background technology

The energy supply of Human Stamina, neuromuscularcontrol ability and bodily form feature etc. also exist significantly race and Individual variation, above-mentioned factor is all affected by gene.Prominent motor capacity is heavily dependent on motion gene genetic Advantage.Progress along with molecular genetics and the application at medicine in field of sports medicine thereof, scientist finds some important health fortune Therbligs matter such as strength, speed and endurance and development potentiality thereof have at a relatively high heritability, and athlete's sensitivity to training is also Affected by inherited genetic factors is the biggest.Inherited genetic factors, as endogenous cause of ill, determines motion talent and development potentiality, the congenital effects of people Space and the limit of development.Research to a large amount of elites shows, congenital heredity advantage accounts for 2/3, and the day after tomorrow trains Effect account for 1/3.Thus, it is found that become the weight of people's autotelic planning growth road with the motion prepotency understanding individual Want reference frame.

Why some people can be easier to obtain brilliant just moving into, it is now recognized that the most relevant with following factor:

1, the difference of ethnic group

Legal collection of illustrative plates (calendar year 2001) research of international human genome shows: the similarity degree of interpersonal gene-code is high Reach 99.99%, the difference of only 0.01%.But this difference of 0.001% just so that individuality has had different on physiology and physical ability Express so that heredity exists between individuality, race, motor capacity difference.The result that sports medical science studies for a long period of time shows, the heart of Black people Blood vessel relatively other ethnic groups are thick, can be high temperature resistant, have an outstanding moment between foot and calf, and buttocks tilting etc., is all them Physical ability feature.It addition, cardio-pulmonary function, muscle types, strength, speed and endurance and development potentiality thereof have at a relatively high heredity Degree, the sensitivity of training is affected by athlete the most to a great extent by genic.

2, sports-related genes and motion key gene

At present there are 206 with the relevant gene that moves, play the motion gene 18 of key effect, but specifically which motion gene Need closely related with Chinese physical ability index is studied further.

There is many institutions conduct motion gene test service in American-European countries has been applied to selection of athletes process, simultaneously Also begin to commercialized development at healthy gene, but owing to ethnic group is different, also not for the detectable of Chinese population exploitation Box.

Summary of the invention

The present invention is directed to the deficiencies in the prior art, a kind of for Chinese motion Dominant Facies correlation gene in order to prepare Detection kit.In order to realize the purpose of the present invention, intend adopting the following technical scheme that

One aspect of the present invention relates to the detection kit of Chinese's motion Dominant Facies correlation gene, it is characterised in that described test kit Including ACE gene the 16th intron insertion or disappearance (I/D) polymorphic detection primer and ACTN3 gene R577X polymorphism inspection Survey primer.

In a preferred embodiment of the present invention, the insertion of described ACE gene the 16th intron or disappearance (I/D) Polymorphic detection primer includes:

Forward: 5 '-CTGGAGACCACTCCCATCCTTTCT-3 '

Reverse: 5 '-GATGTGGCCATCACATTCGTCAGAT-3 '.

In a preferred embodiment of the present invention, described ACTN3 gene R577X polymorphic detection primer includes:

Forward: 5 '-CTG TTGCCTGTGGTAAGTGGG-3 '

Reverse: 5 '-TGGTCACAGTATGCAGGAGGG-3 '.

Another aspect of the present invention relates to the application in detecting object endurance to be measured and/or explosive force of the mentioned reagent box.

Another aspect of the present invention further relates to mentioned reagent box in the reagent of preparation detection Chinese's endurance and/or explosive force Application.

Another aspect of the present invention further relates to the detection method of a kind of Chinese's motion Dominant Facies correlation gene, it is characterised in that bag Include following steps:

1) the peripheric venous blood genomic DNA of object to be measured is extracted；

2) mentioned reagent box PCR is used to expand ACE gene the 16th intron fragment；

3) step 2) PCR primer analysis, analyze the genotype of object to be measured, II type only has 490bp amplified fragments, pure for inserting Mould assembly；DD type only has 190bp, homozygous for disappearance；ID type existing 190bp fragment, has again 490bp fragment, for heterozygous；

4) fragment of mentioned reagent box PCR amplification amplification ACTN3 gene is used

5) step 4) PCR primer is analyzed: use restriction endonuclease DdeI enzyme action pcr amplification product, uses agarose gel electrophoresis analysis Digestion products obtains the genotype of object to be measured, and RR type has 205bp and 86bp two band；XX type has 108bp, 97bp and 86bp tri- Band, for homozygous mutation；The existing 205bp of RX type, has again 108bp, 97bp and 86bp band, for heterozygous mutation.

Test kit of the present invention be exclusively used in detection Chinese motion protogene, can be used for for individual selection or Join a kind of motion or sports events (such as short-distance run/strength exercise or endurance exercise) and be used for evaluating and predicting Exercise performance, the new method of optimization project training plan, it includes evaluating multiple gene or genotype.The test kit of the present invention is also May be used for detecting child, 12 genes relevant to motion advantage and potential of Children and teenager, it was predicted that its talent of moving, Sports item for being engaged in its future provides conductive suggestion.

Detailed description of the invention

If not specified, that technological means used in embodiment is well known to those skilled in the art conventional means.

Embodiment 1:

(1) object of study

1. player group 218 athletes altogether, respectively from track and field team of Zhejiang Province, swimming team and boat team, wherein track and field Athlete 69 (includes 2 national top-notch players and 2 international top-notch players), swimmer 34, paddler 115 (including 8 national top-notch players and 5 international top-notch players).According to research need we they be divide into endurance athletes's group and Speed/fulminant player group, endurance athletes group 129 people altogether include 8 athletes (>=800m), 6 swimming fortune Mobilizing (>=400m) and 115 paddlers, speed/fulminant player group 89 people altogether include 61 athletes (≤400m, shot, javelin, discus, high jump, long-jump) and 28 swimmers (< 400m), wherein endurance athletes organizes male 75 people, women 54 people；Speed/fulminant player group male 43 people, women 46 people.

2. 288 healthy blood donors of matched group, wherein male 149 people, women 139 people.

(2) material, reagent and equipment

1.EDTA anticoagulation: 2ml/ people

2. poba gene group DNA extraction kit (non-centrifugal column type) (Beijing Tian Gen biochemical technology company limited)

3.PCR amplifing reagent

1) PCR primer (sequence sees below)

2) Taq DNA polymerase (5u/ul, Beijing Tian Gen biochemical technology company limited)

3) dNTP(10mM) (Beijing Tian Gen biochemical technology company limited)

4) 10 × PCR Buffer(Beijing Tian Gen biochemical technology company limited)

4. agarose gel electrophoresis reagent

1) agarose (Beijing Tian Gen biochemical technology company limited)

2) storing liquid 5 × TBE(Tris 54g, boric acid 27.5g, EDTA45g add water and are settled to 1000ml, PH8.0), working solution 1 × TBE and 0.5 × TBE.

3) sample-loading buffer (0.25% bromophenol blue, 0.25% dimethylbenzene cyanogen and 40% sucrose)

4) ethidium bromide (storing liquid concentration is 10mg/ml, and working solution concentration is 0.5mg/ml)

5.PCR product column purification system: containing DNA adsorbed film, S1 and W1 solution.The raw work biotechnology in Shanghai services limited public affairs Department.

6. order-checking mixture: BigDye end labelling and order-checking mixture contain 4 kinds of fluorescent labelinies dNTPs, dNTPs, DNA Polymerase and Sequenase.PE company of the U.S..

4 kinds of Terminal fluorescent labels dyestuffs in table 1 BigDye system

In base dyestuff glue image in sequencer map

A dR6G green is green

C Drox redness is blue

G Dr110 blueness black

T Dtamra yellow is red

7. blue dextran/EDTA sample-loading buffer: PE company of the U.S.

8.DNA molecular weight standard

DNA Marker I and DNA Marker II(Beijing Tian Gen biochemical technology company limited)

9. restricted enzyme

HpyF3I(MBI Fermentas)

10. major experimental instrument and equipment

1) PCR instrument: MG 96G type, Hangzhou Lang Ji scientific instrument company limited

2) high speed low temperature centrifugal machine: Megafuge1.0R type, Heraeus company of Germany

3) table model high speed centrifuge: TGL-16B type, Anting Scientific Instrument Factory, Shanghai

4) electronic balance: PB302 type, AB54 type, METTLER TOLEDO company of Switzerland

5) Horizontal electrophoresis tank: Tanon HE-120 Gen Multifunction horizontal electrophoresis tank

6) constant pressure and flow electrophresis apparatus: Tanon EPS300 electrophresis apparatus

7) uv analyzer: sky energy 2500 type gel imaging systems, sky, Shanghai energy

8) DK-8D type electric heating constant temperature tank (Shanghai Medical constant-temperature instrument factory)

9) eddy blending machine: Hua Lida WH-861 type

10) micro sample adding appliance: Gilson company of France

11) refrigerating equipment :-80 DEG C of profound hypothermia refrigerators (Forma Scientific company)

12) nucleic acid-protein analyzer: NanoDrop company of the nd1000(U.S.)

(3) experimental technique

1. human peripheric venous blood extracting genome DNA (RNA isolation kit)

1) in 300 μ l EDTA anticoagulations, 750 μ l cell pyrolysis liquid CL are added, reverse mixing 5 times.

2) 10,000 × g is centrifuged 1 minute.

3) abandon supernatant, centrifuge tube is upside down in clean absorbent paper stop 2 minutes, it is ensured that be deposited in pipe.

4) configuration buffer FG and the mixed liquor (150 μ l buffer FG add 1.5 μ l E.C. 3.4.21.64s) of E.C. 3.4.21.64.

5) adding in centrifuge tube by mixed liquor, vortex mixes to solution without agglomerate immediately.

6) 56 DEG C of water-bath 10min, period reverse mixing is for several times.

7) adding isopropanol 150 μ l, reverse abundant mixing is to thread or tufted genomic DNA occur.

8) 1000,0 × g is centrifuged 3 minutes.Abandon supernatant, centrifuge tube is upside down in clean absorbent paper, it is ensured that be deposited in pipe In.

9) adding 70% ethanol, vortex oscillation 5 seconds, 1000,0 × g is centrifuged 3 minutes.

10) abandon supernatant, centrifuge tube is upside down in clean absorbent paper and stops at least 5 minutes, it is ensured that be deposited in pipe.

11) air dry DNA precipitation until so evaporate clean (at least 5 minutes, it is to avoid overdrying DNA sinks Forming sediment, the DNA being excessively dried is difficult to dissolve).

12) elution buffer TB100 μ l is added, low speed vortex 5 seconds, 65 DEG C of 10 minutes～1 hour dissolving DNA, phases of heating Between reverse mix hydrotropy for several times.

2.DNA concentration measures and preserves

Measure DNA concentration with NanoDrop nucleic acid-protein analyzer nd1000, take 2 μ lDNA solution (being not required to dilution) and be added to loading Platform i.e. can predict concentration at once.The DNA solution of concentration known is diluted to 25ng/ μ l, puts-20 DEG C of preservations.

3. ACE genotype detection

3.1 ACE gene the 16th intron PCR amplifications

3.11 design of primers:

PCR expands ACE gene the 16th intron fragment, and PCR primer sequence is as follows:

Forward: 5 '-CTGGAGACCACTCCCATCCTTTCT-3 '

Reverse: 5 '-GATGTGGCCATCACATTCGTCAGAT-3 '

3.12 PCR reacts

1) reaction system:

DNA profiling 2 μ l(50ng)

10×Buffer 3 μl

25mM MgCl2 3 μl

10mM dNTPs 1 μl

10pmol forward primer 0.8 μ l

10pmol downstream primer 0.8 μ l

Taq archaeal dna polymerase 2 U

Add deionized water to 30 μ l

2) PCR cycle condition: reactant mixture is after 94 DEG C of denaturations 5 minutes, with 94 DEG C of degeneration 60 seconds, anneals 120 seconds for 58 DEG C, 72 DEG C extend 90 seconds, and after totally 35 circulations, last 72 DEG C extend termination amplification in 5 minutes, and PCR amplification uses bright base MG 96G type PCR instrument.

3) PCR primer analysis: take pcr amplification product 5 μ l, adds 1 μ l sample-loading buffer, coagulates in 2% agarose under room temperature Glue constant voltage 120V electrophoresis 20-40 minute, EB dyes 15 minutes, then puts and detects pcr amplification product and spy thereof on uv analyzer The opposite sex, expands again to expanding undesirable person.Crowd has 3 kinds of genotype: II type only has 490bp amplified fragments, pure for inserting Mould assembly；DD type only has 190bp, homozygous for disappearance；ID type existing 190bp fragment, has again 490bp fragment, for heterozygous.

4. ACTN3 genotype detection

The PCR amplification of 4.1 ACTN3 genes

4.11 design of primers:

The fragment of PCR amplification ACTN3 gene, PCR primer sequence is as follows:

Forward: 5 '-CTG TTGCCTGTGGTAAGTGGG-3 '

Reverse: 5 '-TGGTCACAGTATGCAGGAGGG-3 '

Primer is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.

4.12 PCR reacts

1) reaction system:.

DNA profiling 2 μ l(50ng)

10×Buffer 3 μl

25mM MgCl₂ 3 μl

10mM dNTPs 1 μl

10pmol forward primer 0.6 μ l

10pmol downstream primer 0.6 μ l

Taq DNA polymerase 2 U

Add deionized water to 30 μ l

2) PCR cycle condition: reactant mixture is after 94 DEG C of denaturations 5 minutes, with 94 DEG C of degeneration 30 seconds, 58.2 DEG C of annealing 30 Second, 72 DEG C extend 45 seconds, and after totally 35 circulations, last 72 DEG C extend termination amplification in 5 minutes, and PCR amplification uses bright base MG 96G Type PCR instrument.

3) PCR primer analysis: take pcr amplification product 5 μ l, adds 1 μ l sample-loading buffer, coagulates in 2% agarose under room temperature Glue constant voltage 120V electrophoresis 20-40 minute, EB dyes 15 minutes, then puts and detects pcr amplification product and spy thereof on uv analyzer The opposite sex, expands again to expanding undesirable person.

4.2 restricted enzyme action

4.21 endonuclease reaction systems:

PCR primer 10 μ l

Deionized water 16 μ l

10×Buffer Tango 2 μl

Restriction endonuclease DdeI (10U/ μ l) 1 μ l

29 μ l altogether

Add and need to fully mix

4.22 endonuclease reaction temperature and times:

37 DEG C of constant water bath box act on 2～3 hours, puts into 65 DEG C of constant water bath box 20min and terminate reaction.

4.23 digestion products detections:

Use agarose gel electrophoresis.Take digestion products 10 μ l, mix loading, in 3.5% agar with 2 μ l 6 × sample-loading buffers Horizontal strip electrophoresis in sugar gel, 0.5 × tbe buffer liquid, 100V about 1 hour, EB dye 15 minutes, then put and examine on uv analyzer Survey digestion products.Crowd has genotype in 3: RR type has 205bp and 86bp two band；XX type has 108bp, 97bp and 86bp tri- Band, for homozygous mutation；The existing 205bp of RX type, has again 108bp, 97bp and 86bp band, for heterozygous mutation.

5. determined dna sequence

The purification of 5.1 PCR primer: use PCR primer glue to reclaim column purification system.

1) with 1% low melting-point agarose gel electrophoresis separation target DNA fragment, unfertile land cutting as far as possible is containing purpose fragment Gel, puts in 1.5ml centrifuge tube；

2) adding S1 liquid and 1/3 volume isopropanol, mixing, 56 DEG C of water-baths are fully dissolved for 10 minutes；

3) moving liquid to purification column containing DNA adsorbed film, 12000rpm is centrifuged 1 minute, with W1 liquid centrifuge washing twice；

4) adding DNA 30 minutes on deionized water dissolving adsorbed film, 12000rpm is centrifuged 2 minutes and collects DNA lysate.Take on 2 μ l Sample, 1% agarose gel electrophoresis, with DNA molecular amount standard (sky root DNA Marker II) as reference, detection purified fragments Size, purity and concentration.

5.2 sequencing reaction

1) reaction cumulative volume is 20 μ l, Qi Zhonghan:

DNA masterplate 150～300 ng of purification

3.2pmol one side sequencing primer 1 μ l

Sequencing reaction mixture (Mix) 8 μ l

Add deionized water to 20 μ l

2) circular response condition: carry out cycle sequencing reaction in bright base MG 96G type PCR instrument: 96 DEG C 10 seconds, 50 DEG C 5 seconds, 60 DEG C 4 minutes, 25 circulation after, 4 DEG C of preservations.

The purification of 5.3 sequencing reaction products:

1) 20 μ l sequencing reaction product, adds 76% ethanol 80 μ l, fully mixes, and of short duration centrifugal, under room temperature, lucifuge stands 20 points Clock.

2), after 12,000rpm are centrifuged 20 minutes, supernatant is carefully absorbed；

3) add 70% ethanol 250 μ l, fully mix, after 12,000rpm are centrifuged 10 minutes, carefully absorb supernatant；

4) opening centrifugal lid, in PCR instrument, 90 DEG C are dried 2 minutes；

5) add 6 μ l to check order degeneration sample-loading buffers, 96 DEG C of degeneration 3 minutes, be immediately placed in ice bath.

5.4 order-checking electrophoresis: carry out on ABI377 automatic sequencer:

1) in DNA Sequencer software, set sample column, run " Check-plate " Programmable detection electrophoresis glass plate cleaning Degree, " Pre-run program " (1.68KV) carries out prerunning and reaches 56 DEG C to glass plate temperature.

2) loading 2 μ l under prerunning pattern.

3) running " Run " program and carry out electrophoresis (1.68KV, 9 hours), signal collected automatically by software, and electrophoresis is used after terminating DNA Sequencer software combines manually regulation, carries out, by road trace analysis, drawing respective sample to the information in each electrophoresis road Base sequence, prints colored waveform collection of illustrative plates.

6. sequence comparing analysis

With people's ACTN3 gene (gene accession number NM_001104) sequence in GeneBank [www.nibc.nlm.nih.gov] it is Arm's length standard, gained sequence DNA Tool (Ver.5.1) or PC GENE (Ver3.0) software carry out alignment, determine prominent The position become and character.

7. statistical procedures

SPSS13.0 statistical software, allele and genotypic frequency is used to be distributed through genic balance law

A. different types of movement person organizes ACE Genotype distribution

Insertion or disappearance (I/D) polymorphism of being positioned at No. 16 intron of ACE gene can amplify two kinds through PCR method detection DNA fragmentation, a kind of is the DNA fragmentation of 490bp length, referred to as insert type (I type), and another kind is the DNA fragmentation of 190bp length, claims For deletion form (D type), therefore can have 3 kinds of genotype of II, ID and DD in colony, II type only has 490bp fragment, for inserting Type homozygote；DD type only has 190bp fragment, for deletion form homozygote；ID type the most existing 490bp fragment, has again 190bp fragment, For heterozygous.

Table 1 endurance athletes group and fulminant player group ACE genotype men and women's grouped comparison

Note: the value in () represents that this genotype accounts for the percentage ratio of sum.

Relatively compareing height from the studies above result women endurance athletes's II genotypic frequency, DD genotypic frequency is more right According to low, i.e. Chinese women endurance athletes ACE gene polymorphic frequency distribution feature.It addition, comparison crowd ACE gene I/D is polymorphic Frequency distribution has highly significant difference, i.e. in Han nationality general population a high proportion of I allele and II genotype with American-European crowd On the basis of frequency, the specific characteristic of the outstanding endurance athletes drawn in American-European research conclusion is perhaps it is unlikely that in Han nationality In colony, it perhaps it is the other form of expression.

(3) b. different types of movement person organizes ACTN3 Genotype distribution

PCR amplifies the DNA fragmentation of long 290bp, due to mankind's ACTN3 gene R577X polymorphism, may determine that from sequencing result Whether 1747 nucleotide of ACTN3 gene the 16th exon have C to a T site mutation, if occurred without sudden change, are then that RR is pure Close wild type, if this site becomes T, be then XX mutant homozygous type, if existing C has again T, be RX heterozygous.If RR type, Amplification fragment only one of which DdeI restriction enzyme site out, if suddenling change with 1747C to T, then can produce a new DdeI enzyme Cutting site, 3 kinds of ACTN3 genotype pcr amplified fragments are visible after DdeI digestion with restriction enzyme, homozygous mutation XX type warp Can produce 3 kinds of long fragment, respectively 86bp, 97bp and 108bp after enzyme action, RR type only has two kinds of fragments of 86bp and 205bp, RX Type existing 86bp and 205bp fragment, has again 97bp and 108bp fragment, for heterozygous.

Fulminant player group ACTN3 Genotype distribution

In 131 example comparison crowds, ACTN3 genotype is distributed as RR type 36.6% (48/131), and RX type is 48.9% (64/131), XX Type is 14.5% (19/131)；Gene frequency R type is 61.1%, and X-type is 38.9%.89 fulminant athlete's ACTN3 genes Type is distributed as RR type 34.8% (31/89), and RX type is 49.4% (44/89), and XX type is 15.8% (14/89)；Gene frequency R Type is 59.6%, and X-type is 40.4%.It is shown in Table 2 and 3

Table 2 fulminant player group ACTN3 genotype is distributed

Note: the value in () represents that this genotype accounts for the percentage ratio of sum.

Table 3 fulminant player group ACTN3 genotype men and women's grouped comparison

Note: the value in () represents that this genotype accounts for the percentage ratio of sum.

From the above results, women endurance athletes XX genotype proportion and X gene frequency are the most significantly high In matched group, it can be seen that, the X allele of ACTN3 gene is presented with facilitation to the endurance of women endurance athletes.

The above is the preferred embodiments of the present invention, it is noted that come for those skilled in the art Saying, on the premise of without departing from principle of the present invention, it is also possible to make some improvements and modifications, these improvements and modifications also should It is considered as protection scope of the present invention.

Claims (6)

1. the detection kit of Chinese's motion Dominant Facies correlation gene, it is characterised in that include inserting of ACE gene the 16th intron Enter or lack (I/D) polymorphic detection primer and ACTN3 gene R577X polymorphic detection primer.

The detection kit of Chinese's motion Dominant Facies correlation gene the most according to claim 1, it is characterised in that described Insertion or disappearance (I/D) polymorphic detection primer of ACE gene the 16th intron include:

Forward: 5 '-CTGGAGACCACTCCCATCCTTTCT-3 '

Reverse: 5 '-GATGTGGCCATCACATTCGTCAGAT-3 '.

The detection kit of Chinese's motion Dominant Facies correlation gene the most according to claim 1, it is characterised in that described ACTN3 gene R577X polymorphic detection primer includes:

Forward: 5 '-CTG TTGCCTGTGGTAAGTGGG-3 '

Reverse: 5 '-TGGTCACAGTATGCAGGAGGG-3 '.

4. mentioned reagent box application in detecting object endurance to be measured and/or explosive force, object to be measured is women.

5. mentioned reagent box application in the reagent of preparation detection Chinese's endurance and/or explosive force, object to be measured is women.

6. the detection method of Chinese's motion Dominant Facies correlation gene, it is characterised in that comprise the steps:

1) the peripheric venous blood genomic DNA of object to be measured is extracted；

2) mentioned reagent box PCR is used to expand ACE gene the 16th intron fragment；

3) step 2) PCR primer analysis, analyze the genotype of object to be measured, II type only has 490bp amplified fragments, pure for inserting Mould assembly；DD type only has 190bp, homozygous for disappearance；ID type existing 190bp fragment, has again 490bp fragment, for heterozygous；

4) fragment of mentioned reagent box PCR amplification ACTN3 gene is used；

5) step 4) PCR primer is analyzed: use restriction endonuclease DdeI enzyme action pcr amplification product, uses agarose gel electrophoresis analysis Digestion products obtains the genotype of object to be measured, and RR type has 205bp and 86bp two band；XX type has 108bp, 97bp and 86bp tri- Band, for homozygous mutation；The existing 205bp of RX type, has again 108bp, 97bp and 86bp band, for heterozygous mutation；

Preferably, described object to be measured is women.