CN107385036A

CN107385036A - A kind of detection primer group, kit and the application in ARR3 genes T239C, C298T and C893A sites

Info

Publication number: CN107385036A
Application number: CN201710598012.2A
Authority: CN
Inventors: 张清炯; 肖学珊; 黎仕强; 贾小云; 郭向明
Original assignee: Zhongshan Ophthalmic Center
Current assignee: Zhongshan Ophthalmic Center
Priority date: 2017-07-20
Filing date: 2017-07-20
Publication date: 2017-11-24
Anticipated expiration: 2037-07-20
Also published as: CN107385036B

Abstract

The invention discloses a kind of detection primer group, kit and the application in ARR3 genes T239C, C298T and C893A sites.Principle of the present invention based on Sanger sequencings, the characteristics of sensitiveness, stability and accuracy with height, the mutational site of three cause high myopias of ARR3 genes can be detected simultaneously, early screening, auxiliary diagnosis for high myopia, implement early prevention for patient and treatment provides foundation, there is good market application foreground.

Description

A kind of detection primer group in ARR3 genes T239C, C298T and C893A sites, reagent Box and application

Technical field：

The invention belongs to biological technical field, and in particular to a kind of inspection in ARR3 genes T239C, C298T and C893A sites Survey primer sets, kit and application.

Background technology：

Myopia is to endanger the common cause of eyesight, wherein, high myopia (myopia of the diopter more than -6.00D) is concurrent Disease (glaucoma, cataract, vitreous opacity, detachment of retina etc.) is the second largest reason of blinding；The myopia of Chinese is ill Rate is far above other races, and involvement all the life.It is severeer, with the propulsion of urbanization and the transformation of life style, myopia The incidence of disease also increasing year by year, inhabit the asian population of developed area more so.A large amount of evidences show high myopia with Hereditary closely related, Familial Occurrence high myopia, especially preschool existing early hair high myopia, by such environmental effects Small, mostly caused by gene mutation, this kind of patient's myopia degree progressive increase, eye ground choroidopathy adds year by year Weight, and be handed down from age to age, bring heavy psychology and financial burden to patient.If the base that causes a disease is carried out to this kind of high myopia It is its early diagnosis and the basis prevented and treated because of abrupt climatic change.

The current existing Disease-causing gene detection kit to early onset high myopia, only the six of ZNF644 genes cause The detection (CN102732607A) in sick site, but this six pathogenic sites only detect in very few patient, and absolutely mostly The pathogenic mutation of several early onset high myopias is failed to understand, it is also necessary to carries out abrupt climatic change to more Disease-causing genes.Therefore Find the mutational site that can more cause early onset high myopia and develop into detection kit, by early onset high myopia Accurate diagnosis and treatment are significant.

The content of the invention：

It is an object of the invention to provide ARR3 genes T239C, C298T that a species specificity is good, high sensitivity, stability are strong With detection primer group, kit and the application in C893A sites.

First purpose of the present invention is to provide a kind of detection primer in ARR3 genes T239C, C298T and C893A sites Group, described detection primer group are as follows：

For ARR3 gene T239C and C298T sites：

ARR3-1F：5 '-GCCAGCCTTCGATCATATTACAT-3 ' (as shown in SEQ ID NO.1)；

ARR3-1R：5 '-TGGCGATGAGGCTGTGGAGGTTA-3 ' (as shown in SEQ ID NO.2)；

For ARR3 gene C 893A sites：

ARR3-2F：5 '-AGGATTTCCCACCAGAACTAAT-3 ' (as shown in SEQ ID NO.3)；

ARR3-2R：5 '-GGCTTTACTGAACATGAAACCT-3 ' (as shown in SEQ ID NO.4).

Second object of the present invention is to provide a kind of ARR3 genes T239C and C298T sites and/or C893A sites Detection kit, including it is above-mentioned for the upstream and downstream primer in ARR3 gene T239C and C298T sites and/or for ARR3 bases Because of the upstream and downstream primer in C893A sites.

It is preferred that described detection kit also includes 2*Master Mix, ddH₂O, positive quality control product and negative quality-control product.

It is preferred that described positive quality control product includes containing the DNA of sequence shown in SEQ ID NO.5, contains SEQ ID The DNA of sequence shown in NO.6 and the DNA containing sequence shown in SEQ ID NO.7.

Third object of the present invention is to provide a kind of detection method in ARR3 genes T239C, C298T and C893A sites, Comprise the following steps：

(1) genomic DNA of testing sample is extracted；

(2) respectively with above-mentioned for the upstream and downstream primer in ARR3 gene T239C and C298T sites and for ARR3 genes The genomic DNA that the upstream and downstream primer in C893A sites is extracted to step (1) enters performing PCR amplification；

(3) pcr amplification product is sequenced, be then compared with wild type ARR3 gene orders, it is determined whether deposit In gene mutation site.

The method of described sequencing is preferably Sanger PCR sequencing PCRs.

Principle is sequenced in Sanger, and DNA profiling exists in archaeal dna polymerase, primer, four kinds of deoxynucleotide triphosphoric acids (dNTP) Under when being replicated, introduce the dideoxyribonucleoside of four kinds of fluorescent dyes marks according to certain ratio respectively in reaction system Triphosphoric acid (ddNTP).Because ddNTP lacks 3 '-OH groups required for extension, when the end of ddNTP incorporation chains, the chain is just It can stop extending.A series of DNA fragmentation using ddNTP as 3 ' ends that length do not wait just is generated in so per tube reaction system. After reaction terminating, gel electrophoresis is carried out in capillary to separate DNA fragmentation different in size, adjacent fragment length difference one Individual base, the mobility in Capillary Electrophoresis is just different, and laser detector can detect one by one to fluorescence molecule, and Different fluorescence can be changed into DNA sequence dna by synchronous imaging on CCD camera, analysis software automatically, so as to reach the mesh of DNA sequencing 's.

Described PCR amplifications, its reaction system is preferably 20 μ L, includes genomic DNA 60ng, 2*Master Mix 10 μ L, 10 μM of sense primers 0.3 μ L, 10 μM of anti-sense primers 0.3 μ L, ddH₂O complements to 20 μ L.

Described PCR amplifications, its response procedures are preferably：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 45s, repeats 30 circulations；72 DEG C of extension 5min.

Fourth object of the present invention is to provide above-mentioned detection primer group or above-mentioned detection kit in the early hair of detection Application in property high myopia.

The present invention devises a set of detection primer group for tri- mutational sites of ARR3 genes T239C, C298T and C893A, Develop a kit comprising detection primer group and the detection method using the kit.This kit is using classics Sanger dideoxy chain-termination PCR sequencing PCRs, this method result is accurate, visual, repeatability height directly perceived, and high specificity and sensitivity Height, it is the goldstandard of gene diagnosis.

Principle of the present invention based on Sanger sequencings, has the characteristics of sensitiveness of height, stability and accuracy, can be with The mutational site of three cause high myopias of ARR3 genes, early screening, auxiliary diagnosis for high myopia, to suffer from are detected simultaneously Person implements early prevention and treatment provides foundation, has good market application foreground.

Embodiment：

Following examples are to further explanation of the invention, rather than limitation of the present invention.

Embodiment 1：

1st, the design of specific primer

According to (the GeneBank Serial No. Gene ID of ARR3 genes in ncbi database:407；NC_000023)T239C (i.e. shown in SEQ ID NO.5 sequence from 5 ' end rise the 202nd bit base be T/C), C298T (i.e. shown in SEQ ID NO.6 sequence oneself 5 ' to have held the 261st bit base be C/T) and C893A (sequence the 293rd bit base from 5 ' ends is C/A i.e. shown in SEQ ID NO.7) The upstream and downstream sequences Design specific primer in site, designed specific primer are as shown in table 1.

The specific primer sequence of table 1

2nd, extracting genome DNA

200 normal persons are extracted respectively using phenol/chloroform method or isolation kit method and 200 early onset high myopias are suffered from Person's peripheral white blood cells genomic DNA.Comprise the following steps that：

2.1 add 200 μ L Proteinase K (20mg/mL) solution to 15mL centrifuge tubes；

2.2 processing materials：Blood sample is extracted, is directly added into 3mL blood samples, is mixed；

2.3 add 3.6mL buffer solution GE into the centrifuge tube equipped with blood sample, and vibration 30sec is mixed；

2.4 65 DEG C of placement 10min, every 3min vibrations once, to help cracking.Brief centrifugation is to collect cap wall The globule；

2.5 add 3mL absolute ethyl alcohols into sample, mix, are now likely to occur flocculent deposit；

2.6 are transferred to the half of previous step resulting solution and flocculent deposit in one adsorption column CB5 that (adsorption column is put into In 15mL collecting pipes), 3,000rpm (~1,850 × g) centrifugation 3min, waste liquid is outwelled, adsorption column CB5 is put back in collecting pipe；

2.7 are transferred to the remaining solution of step 2.6 in same adsorption column again, and repeat step 2.6 operates；

2.8 into adsorption column CB5 add 2mL buffer solutions GD (please first checked whether before use and added absolute ethyl alcohol), 5, 000rpm (~4,500 × g) centrifuges 1min, outwells waste liquid, adsorption column CB5 is put back in collecting pipe；

2.9 into adsorption column CB5 add 2mL buffer solutions PW (please first checked whether before use and added absolute ethyl alcohol), 5, 000rpm (~4,500 × g) centrifuges 1min, outwells waste liquid, adsorption column CB5 is put back in collecting pipe, the DNA of collection is standby.

3rd, plasmid is prepared

Sequencing is tested after SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7 are cloned on PMD19-T carriers respectively Clone is demonstrate,proved, mutant plasmids PMD19-T1, mutant plasmids PMD19-T2 and mutant plasmids PMD19-T3 are obtained, by SEQ Sequence verification clone, obtains wild plasmid after ID NO.8 and SEQ ID NO.9 are cloned on PMD19-T carriers respectively PMD19-T4 and wild plasmid PMD19-T5；Extract DNA.

4th, PCR is expanded

4.1 genomic DNAs extracted using step 2 is templates, respectively with the primer pair ARR3-1F/R and ARR3- shown in table 1 2F/R enters performing PCR amplification.Positive control is respectively mutant plasmids PMD19-T1 and mutant plasmids PMD19-T2.Negative control For PMD19-T.Using 2 μ L pure water as template, make blank control.

4.2PCR reaction system：Total system is 20 μ L, includes genomic DNA 2 the μ L, 2*Master that concentration is 30ng/ μ L Mix 10 μ L, 10 μM of sense primers 0.3 μ L, 10 μM of anti-sense primers 0.3 μ L, ddH₂O 7.4μL；PCR response procedures：94 DEG C of pre- changes Property 5min；94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 45s, repeat 30 circulations；72 DEG C of extension 5min.

4.3PCR amplified productions detect：3 μ L pcr amplification product is taken, (contains 0.5 μ g/mL in 20g/L Ago-Gels EB), 50V electrophoresis 45-60min, result is directly observed under uviol lamp：Expanded by primer of ARR3-1F/R, 200 normal The genomic DNA and positive control of people and 200 early onset high myopias amplify a 873bp or so band； Expanded by primer of ARR3-2F/R, the genomic DNA and sun of 200 normal persons and 200 early onset high myopias Property control amplify a 400bp or so band.

4.4PCR amplified productions purify：

Operating procedure：Absolute ethyl alcohol is please first added in rinsing liquid PW before, addition volume refer to the label on bottle. All centrifugation steps are to be centrifuged at room temperature using desk centrifuge.

4.4.1 column equilibration step：Into adsorption column CB3, (adsorption column is put into collecting pipe) adds 500 μ L equilibrium liquid BL, 12,000rpm (~13,400 × g) centrifuge 1min, outwell the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe. (pillar that please be treated on the use same day).

4.4.2 estimate the volume of PCR reaction solutions, add the combination liquid PB of 5 times of volumes thereto, fully mix.

4.4.3 previous step resulting solution is added in an adsorption column CB3 (adsorption column is put into collecting pipe), room temperature is placed 2min, 12,000rpm (~13,400 × g) centrifuge 30-60sec, outwell the waste liquid in collecting pipe, adsorption column CB3 is put into receipts In collector.Pay attention to：Absorption column volume is 800 μ L, if sample volume is more than 800 μ L and can be added portionwise.

4.4.4 600 μ L rinsing liquids PW are added into adsorption column CB3 and stand 2-5min, 12,000rpm (~13,400 × g) 30-60sec is centrifuged, the waste liquid in collecting pipe is outwelled, adsorption column CB3 is put into collecting pipe.

4.4.5 repeat step 4.4.4.

4.4.6 centrifugal adsorbing column CB3 is put back in collecting pipe, 12,000rpm (~13,400 × g) centrifugation 2min, as far as possible Remove rinsing liquid.Adsorption column is placed in into room temperature to place several minutes, thoroughly dried, to prevent the rinsing liquid of residual from influenceing in next step Experiment.Collect purified product.

4.4.7 sequencing reaction：Be separately added into certain sequence into reaction mixture purified DNA cloning Product samples, Each 2 μ L of positive control, negative control.

PCR response procedures are sequenced：94 DEG C, pre-degeneration 5min；94 DEG C of denaturation 10s, 61 DEG C of annealing 4min, repeat 30 and follow Ring.

4.5 sequencing amplified production purifying：

4.5.1 the μ L of sequencing reaction amplified production 5 and the μ L of 70% absolute ethyl alcohol 35,4,000rpm centrifugations are added in 96 orifice plates 30min, 96 orifice plates 500rpm, short 30sec in turn.

4.5.2 room temperature is placed 40min and dried.

Machine is sequenced on 4.6：

Upper this preparation of press proof：Sample after drying adds the μ L of Hi-Di deionized formamides 10,95 DEG C of 5min denaturation, then horse On be placed in cooled on ice 5min.Then it is put on the sample plane pedestal of sequenator and is sequenced.

Sequencing result is analyzed：Initial data is exported, " DNAStar " software is used, sequencing sequence and original normal sequence (Gene ID:407NC_000023, i.e. wild type ARR3 gene orders) it is compared.

As a result show：T239C, C298T and C893A site of 200 normal persons is not undergone mutation, is feminine gender；200 1 patient T239C site mutation, 1 C298T site mutation and 1 patient C893A site in name early onset high myopia Mutation, we so that the family member of three above patient is analyzed with identical method, find：T239C familys 8 into 4 in member containing this site mutation are high myopia, in addition 4 it is normal per capita without this site mutation；C298T 10 in 12 members of family carry this site mutation is high myopia, in addition 2 it is normal per capita without this position Point mutation；15 in 30 members of C893A familys carry this site mutation is high myopia, in addition 15 it is normal This site mutation is not carried per capita.Illustrate mutation and the early onset high myopia in these three sites of T239C, C298T and C893A It is significantly correlated.

Embodiment 2：

1st, sensitivity technique

1.1 plasmids dilute

Respectively by mutant plasmids PMD19-T1, PMD19-T2 and PMD19-T3, wild plasmid PMD19-T4, PMD19- T5, final concentration 5ng/ μ L are diluted to, then by the corresponding wild plasmid of mutant plasmids by volume 0.2:9.8、 0.5:9.5、1:9、1.5:8.5、2:8、3:7 and 4:6 are mixed, be configured to be mutated abundance be 2%, 5%, 10%, 15%, 20%th, 30% and 40% positive sample (table 2).Using the positive sample of different mutation abundance as template, according to the institute of embodiment 1 The method stated enters performing PCR and expands and be sequenced.As a result it is as shown in table 3.Judge that PRELIMINARY RESULTS shows this hair with reference to sequencer map and table 3 Bright detection kit (Sanger PCR sequencing PCRs) detection sensitivity is that mutation abundance is 10%.

The plasmid information of the mutant plasmid of table 2

The sensitivity technique result of table 3 (forward and reverse test)

Mutant plasmid is mutated abundance

2%

5%

10%

15%

20%

30%

40%

T239C

-/-

+/+

C298T

-/-

+/+

C893A

-/-

+/+

1.2 sensitivity are verified

Criterion of acceptability：The positive sample of detection mutation abundance 10% 10 times, at least 9 testing results meet Monitoring lower-cut, I.e. recall rate is more than or equal to 90%.

Verify the detection sensitivity of mutant abundance 10%

Take mutant plasmid PMD19-T1/PMD19-T4, PMD19-T2/ of the obtained mutation abundance 10% of step 1.1 PMD19-T4, PMD19-T3/PMD19-T5 carry out gene mutation with the detection kit (Sanger PCR sequencing PCRs) of the present invention to it Detection.10 repetitions are sequenced, and recall rate is 100% (table 4).Sensitivity is verified.

The sensitivity of table 4 checking testing result (mutation abundance 10%)

Detect sample (mutation abundance 10%)	Detect number	Detect number
			T239C	10	10
C298T	10	10
			C893A	10	10

2nd, specific test：

2.1 specificity verification methods：By the primer pair ARR3-1F/R shown in table 1 in people, macaque, wild boar, hog and white rhinoceros Matched in cow genome group sequence with electronic simulation PCR.By the primer pair ARR3-2F/R shown in table 1 in people, wild boar, hog Matched with white rhinoceros genome sequence with electronic simulation PCR.

As a result show：ARR3-1F/R primers can generate specific product in human genome sequence, macaque, wild boar, Without generation amplified production in hog and white rhinoceros genome sequence；ARR3-2F/R primers can give birth in human genome sequence Into specific product, without generation amplified production in macaque, wild boar, hog and white rhinoceros genome sequence.Therefore the present invention Detection kit there is the specificity of height.

3rd, reperformance test：

3.1 reperformance test methods：By mutant plasmids PMD19-T1, PMD19-T2 and PMD19-T3 according to step 2.1 Method be diluted to 10%, 30% two mutation abundance sample be used as template, according to the method for embodiment 1 enter performing PCR expand, 10 repetitions are sequenced, and calculate its positive findings repetitive rate (being shown in Table 5).

The different mutation abundance samples in two kinds of 3 site of table 5ARR3 genes repeat sequencing result

3.2 conclusion：Comprehensive sequencer map and table 5 show, the site 10% of ARR3 genes 3 and each 10 times of 30% two kind of mutation abundance It is 100% to repeat sequencing positive findings repetitive rate, and the amplimer and the detection of PCR reaction conditions are reproducible, and can contrast The sample of mutation abundance 10% realizes stable detection.

Sequence table

<110>Zhongshan Ophthalmic Center, Sun Yat-sen University

<120>A kind of detection primer group, kit and the application in ARR3 genes T239C, C298T and C893A sites

<160> 9

<210> 1

<211> 23

<212> DNA

<213>Artificial sequence

<400> 1

gccagccttc gatcatatta cat 23

<210> 2

<211> 23

<212> DNA

<213>Artificial sequence

<400> 2

tggcgatgag gctgtggagg tta 23

<210> 3

<211> 22

<212> DNA

<213>Artificial sequence

<400> 3

aggatttccc accagaacta at 22

<210> 4

<211> 22

<212> DNA

<213>Artificial sequence

<400> 4

ggctttactg aacatgaaac ct 22

<210> 5

<211> 873

<212> DNA

<213>People

<400> 5

gccagccttc gatcatatta catcacctct ctgtgtcttc ttgacccagt taagattccc 60

cttcccactc aggcctgaca gaccactctc ttcctcctat gcctgcagtg tttgtcatgt 120

tgacatgtgc ctttcgctat ggccgtgatg acttggaagt gattggtctg acgttccgaa 180

aagatctgta tgtgcagacc ccgcaagtgg tcccagctga atccagcagc cctcaggggc 240

ccctcacagt cctacaggag cgactactgc acaagctagg ggacaatgcc taccccttta 300

ccctgcaggt actgacccca agccctgggc aggcaaggtc tccagggaag attaagagag 360

gaagctcaag aaggacaaca agggagaggg ttccccattt cttttgtatt tgtttgtatt 420

cttttctact tcttttctga tctccttttt gtcccctaga tggtgaccaa cctgccctgt 480

tctgtgacac tgcagccagg tcctgaagat gcaggaaagg tgaggactgg gttctaagaa 540

agagggatag gtagtgccag ggaagaggaa cagtgggaca gtcaagactg gagaaatgga 600

cagctaagga aagaggtcag gcattttctt ataggcccat gaggaaggga ggacagcact 660

ggaaatgagc tctctttgcc cttgtccctt tacagccctg tgggattgac tttgaagtga 720

agagtttctg tgctgaaaac ccagaggaga cagtctccaa gaggtattct ttggttgtcc 780

ccaaaaatcc ctgcctccag cctctgccag gaaacagatc ctgctctcat gacagaaata 840

gccccacttc taacctccac agcctcatcg cca 873

<210> 6

<211> 873

<212> DNA

<213>People

<400> 6

gccagccttc gatcatatta catcacctct ctgtgtcttc ttgacccagt taagattccc 60

cttcccactc aggcctgaca gaccactctc ttcctcctat gcctgcagtg tttgtcatgt 120

tgacatgtgc ctttcgctat ggccgtgatg acttggaagt gattggtctg acgttccgaa 180

aagatctgta tgtgcagacc ctgcaagtgg tcccagctga atccagcagc cctcaggggc 240

ccctcacagt cctacaggag tgactactgc acaagctagg ggacaatgcc taccccttta 300

ccctgcaggt actgacccca agccctgggc aggcaaggtc tccagggaag attaagagag 360

gaagctcaag aaggacaaca agggagaggg ttccccattt cttttgtatt tgtttgtatt 420

cttttctact tcttttctga tctccttttt gtcccctaga tggtgaccaa cctgccctgt 480

tctgtgacac tgcagccagg tcctgaagat gcaggaaagg tgaggactgg gttctaagaa 540

agagggatag gtagtgccag ggaagaggaa cagtgggaca gtcaagactg gagaaatgga 600

cagctaagga aagaggtcag gcattttctt ataggcccat gaggaaggga ggacagcact 660

ggaaatgagc tctctttgcc cttgtccctt tacagccctg tgggattgac tttgaagtga 720

agagtttctg tgctgaaaac ccagaggaga cagtctccaa gaggtattct ttggttgtcc 780

ccaaaaatcc ctgcctccag cctctgccag gaaacagatc ctgctctcat gacagaaata 840

gccccacttc taacctccac agcctcatcg cca 873

<210> 7

<211> 400

<212> DNA

<213>People

<400> 7

aggatttccc accagaacta atttctccca gtcttcagtt aaacatgtta cctctcttgg 60

atactccagt aggttcttgt ataatcttgt acaagatact cttgggagta aaagtatgct 120

tagtcagcaa gcccagccta aattcttctc ttgttcttct tttgtaggga gactgtagct 180

gctaattcca gcttctccca gagctttgca gtaaccccaa tcctggctgc cagctgccag 240

aaacggggcc tggcactgga tggcaaactt aagcatgaag ataccaacct ggactctagc 300

acaatgtaag ctcaaatata actctcagcc tccatttcct acccctcaac cattccacat 360

gctacattta cagctctgag gtttcatgtt cagtaaagcc 400

<210> 8

<211> 873

<212> DNA

<213>People

<400> 8

gccagccttc gatcatatta catcacctct ctgtgtcttc ttgacccagt taagattccc 60

cttcccactc aggcctgaca gaccactctc ttcctcctat gcctgcagtg tttgtcatgt 120

tgacatgtgc ctttcgctat ggccgtgatg acttggaagt gattggtctg acgttccgaa 180

aagatctgta tgtgcagacc ctgcaagtgg tcccagctga atccagcagc cctcaggggc 240

ccctcacagt cctacaggag cgactactgc acaagctagg ggacaatgcc taccccttta 300

ccctgcaggt actgacccca agccctgggc aggcaaggtc tccagggaag attaagagag 360

gaagctcaag aaggacaaca agggagaggg ttccccattt cttttgtatt tgtttgtatt 420

cttttctact tcttttctga tctccttttt gtcccctaga tggtgaccaa cctgccctgt 480

tctgtgacac tgcagccagg tcctgaagat gcaggaaagg tgaggactgg gttctaagaa 540

agagggatag gtagtgccag ggaagaggaa cagtgggaca gtcaagactg gagaaatgga 600

cagctaagga aagaggtcag gcattttctt ataggcccat gaggaaggga ggacagcact 660

ggaaatgagc tctctttgcc cttgtccctt tacagccctg tgggattgac tttgaagtga 720

agagtttctg tgctgaaaac ccagaggaga cagtctccaa gaggtattct ttggttgtcc 780

ccaaaaatcc ctgcctccag cctctgccag gaaacagatc ctgctctcat gacagaaata 840

gccccacttc taacctccac agcctcatcg cca 873

<210> 9

<211> 400

<212> DNA

<213>People

<400> 9

aggatttccc accagaacta atttctccca gtcttcagtt aaacatgtta cctctcttgg 60

atactccagt aggttcttgt ataatcttgt acaagatact cttgggagta aaagtatgct 120

tagtcagcaa gcccagccta aattcttctc ttgttcttct tttgtaggga gactgtagct 180

gctaattcca gcttctccca gagctttgca gtaaccccaa tcctggctgc cagctgccag 240

aaacggggcc tggcactgga tggcaaactt aagcatgaag ataccaacct ggcctctagc 300

acaatgtaag ctcaaatata actctcagcc tccatttcct acccctcaac cattccacat 360

gctacattta cagctctgag gtttcatgtt cagtaaagcc 400

Claims

1. a kind of detection primer group in ARR3 genes T239C, C298T and C893A sites, it is characterised in that described detection is drawn Thing group is as follows：

For ARR3 gene T239C and C298T sites：

ARR3-1F：5’-GCCAGCCTTCGATCATATTACAT-3’；

ARR3-1R：5’-TGGCGATGAGGCTGTGGAGGTTA-3’；

For ARR3 gene C 893A sites：

ARR3-2F：5’-AGGATTTCCCACCAGAACTAAT-3’；

ARR3-2R：5’-GGCTTTACTGAACATGAAACCT-3’.

2. a kind of ARR3 genes T239C and C298T sites and/or the detection kit in C893A sites, it is characterised in that including Described in claim 1 for the upstream and downstream primer in ARR3 gene T239C and C298T sites and/or for ARR3 gene Cs 893A The upstream and downstream primer in site.

3. detection kit according to claim 2, it is characterised in that also including 2*Master Mix, ddH₂O, positive matter Control product and negative quality-control product.

4. detection kit according to claim 3, it is characterised in that described positive quality control product includes containing SEQ ID The DNA of sequence shown in NO.5, containing the DNA of sequence shown in SEQ ID NO.6 and contain sequence shown in SEQ ID NO.7 The DNA of row.

5. a kind of detection method in ARR3 genes T239C, C298T and C893A sites, it is characterised in that comprise the following steps：

(1) genomic DNA of testing sample is extracted；

(2) respectively with described in claim 1 for ARR3 gene T239C and C298T sites upstream and downstream primer and be directed to The genomic DNA that the upstream and downstream primer in ARR3 gene C 893A sites is extracted to step (1) enters performing PCR amplification；

(3) pcr amplification product is sequenced, be then compared with wild type ARR3 gene orders, it is determined whether base be present Because of mutational site.

6. detection method according to claim 5, it is characterised in that the method for described sequencing is Sanger PCR sequencing PCRs.

7. detection method according to claim 5, it is characterised in that described PCR amplifications, its reaction system is 20 μ L, Comprising genomic DNA 60ng, 2*Master Mix 10 μ L, 10 μM of sense primers 0.3 μ L, 10 μM of μ L of anti-sense primer 0.3, ddH₂O complements to 20 μ L.

8. detection method according to claim 5, it is characterised in that described PCR amplifications, its response procedures are：94℃ Pre-degeneration 5min；94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 45s, repeat 30 circulations；72 DEG C of extension 5min.

9. the detection kit described in detection primer group or claim 2 described in claim 1 is highly near in detection early onset Application depending in.