CN103060434B - Reagent and method for predicting susceptibility to prostate cancer - Google Patents

Abstract

The invention discloses a reagent and a method for predicting susceptibility to prostate cancer, belonging to the field of biotechnology. The reagent and the method can be used for predicting susceptibility of a subject to prostate cancer by extracting the genomic deoxyribonucleic acid (DNA) of host cells and testing the genotype of the rs1321366 site on the eleventh intron of RFX 6 gene of the subject; when the genotype of the rs1321366 on the eleventh intron of the RFX 6 gene is GG, the susceptibility of the subject to prostate cancer is the highest; and when the genotype of the rs1321366 is AG or AA, the susceptibility of the subject to prostate cancer is lower. The reagent and the method have the advantages that the correlation between rs1321366 gene polymorphic site and prostate cancer is expounded for the first time; the method for predicting the susceptibility to prostate cancer is provided; and the method can be used for early prevention diagnosis and auxiliary diagnosis of prostate cancer, and is also used for research and development of new medicines.

Description

A kind of reagent and method of predicting susceptibility to prostate cancer

Technical field

The present invention relates to a kind of reagent and method of predicting susceptibility to prostate cancer, say more specifically by measuring with relating to prostate cancers because rs1321366 loci polymorphism prediction experimenter on the intron of RFX6 is for the susceptibility of prostate cancer, the method can be used for auxiliary diagnosis and the new drug development of disease, belongs to biological technical field.

Background technology

Prostate cancer (prostate cancer, PCa) is to occur in modal malignant tumour in male reproductive system.The U.S., after 1975-2006 cancer morbidity is added up, estimates that PCa new cases in 2010 are 217,730 examples, occupy the male sex and swell well in knurl first, the death toll of PCa is estimated as 32,050, occupies the second (Jemal A in male tumor mortality ratio, Siegel R, Xu J, Ward E.Cancer statistics, 2010.CA Cancer J Clin, 2011,60 (5): 277-300.).Age is an important risk factor of PCa, and the basic seldom morbidity that 45 years old age is following, increases with the growth at age.American Cancer Society statistics, 40 years old following male sex PCa sickness rate is in 1/8499,40-59 year male sex to be 1/38, in 60-69 year male sex, is more than 1/15,70 years old in the male sex, to be 1/8.Along with our national aging population, the sickness rate of PCa rises just year by year in China, upper seamark sickness rate from 1973～1975 years 1.8/10 ten thousand rise to the 5.5/10 ten thousand (Liu Zhenwei of 1997～1999 years, Xiang Yongbing, Zhang Wei. 1973～1999 years PCa analysis of pathogenetic tendencies in Areas in Shanghai City. China Health statistics, 2003, 20 (6): 335-337.), the sickness rate of the PCa of part counties and cities, southern areas, Guizhou Province from 1994～1998 years 1.72/10 ten thousand rise to 2004～2008 years 4.28/10 ten thousand, become the common problem (Huang Guijun that threatens China's men's health, Lan Zejun, Quan Xin. the PCa incidence trend research in 1994～2008 years of part counties and cities, southern areas, Guizhou Province. modern preventive medicine, 2011, 37 (116): 3012-3017.).

Carry out at present the research of PCa inherited pathogenic factor, the checkings that adopt in extensive genome-wide association study and the many crowds of small sample more, and multiple PCa susceptible SNPs of GWAS qualification have been replicated definite in multiple different crowds, proved SNPs as the association analysis method of genomic marker the validity in PCa genetic research.SNPs refers to the DNA sequence polymorphism that in chromogene group level, single nucleotide diversity causes, frequency in crowd needs >1%, SNPs is biallelic marker, during changing, this single base has 70.1% for the conversion between homotype base: as G/A or T/C, 29.1% for occurring in the transversion between purine and pyrimidine.The 80-90% that SNPs has comprised known polymorphism is modal heritable variation.

Because the selective pressure of existence causes the distribution of SNP in term single gene and whole genome to be ununiformity.SNPs is 4 times of coding region in the quantity of gene non-coding region, and sum can reach 300 ten thousand.SNPs taking its density high (average every 1kb just has 1), representative strong (SNPs that is arranged in gene inside may directly affect protein structure or expression level), genetic stability good (with microsatellite polymorphism comparatively speaking), be easy to the features such as automated analysis (because of SNPs crowd as biallelic marker, can be simply with " +/-or 1/0 " direct somatotype) and become good genetic marker.

Rfx6(regulatory factor X6)---belong to transcription factor RFX(regulatory factor X-box binding) family.Be positioned at chr15(54166958-54222377), 19 exons, proteins encoded length 1281, a hypotype, has tri-structural domains of B, C and D, mainly expresses at pancreas, lower than expression level with the RFX of family with other, in the development of pancreas and the adjusting of function, be crucial transcriptional.RFX6 and RFX2 and RFX3 direct effect, and the latter two are expressed and play a role at pancreas.

In pancreas, Rfx6 is positioned at short endocrine factor Neurog3 downstream, and the upstream of other multiple islet transcription factors plays a role in islet cells differentiation.The sudden change of these two kinds of genes shows similar and different phenotype.Mankind RFX6 sudden change, can cause intractable diarrhoea and diabetes, intestinal occlusion and anomalies of biliary system, neonatal diabetes.Fully understanding of Rfx6 contributed to illustrate the formation of pancreas islet and β cell, the pathogenic process of diabetes.

DNA-binding albumen RFX6 plays a significant role in the pathology of newborn hemochromatosis, and the dependency of hemochromatosis HFE gene and prostate cancer has been given prominence to the pathology of hemochromatosis and the interaction of prostate cancer.

Near site GPRC6A and RFX6 gene, constriction is to SNPs and the prostate cancer significant correlation in gene RFX6 region, than in initial GWAS with the related locus GPRC6A/RFX6 of prostate cancer, RFX6 genovariation may be responsive site more preferably.

2010, Takata R etc. identified the rs339331 and the PCa risk association that are positioned on RFX6 gene, and after this, checking is copied in this site in European crowd and Chinese population.Rs339331 is positioned at RFX6 gene intron 4, with 59 tag SNPs covering 580kb region (Chr.6:117.0 – 117.6Mb) mapping analysis, it is positioned at the associated section of an about 200kb, this region is contained two gene: GPRC6A, RFX6 is in conjunction with the above analysis to RFX6 function, we infer that RFX6 gene is PCa tumor susceptibility gene and the rs1321366 that has selected to be positioned at it, in 273 PCa patients and 606 contrast crowds, after somatotype, carry out association analysis, result has determined that rs1321366 is associated with Chinese population PCa.

Rs1321366 is set to 117 karyomit(e) is upper, 347,285, be positioned on the 11 intron of RFX6, this exon has 1013bp, rs1321366 is the SNP site apart from initiating terminal 138, and intron is played an important role at alternative splicing, and therefore a gene can produce multiple different protein.Through query and search, so far, RFX6 gene rs1321366 and PCa risk yet there are no report.

Summary of the invention

Main purpose of the present invention is to provide a kind of method that detects susceptibility to prostate cancer gene.

Second object of the present invention is to provide a kind of reagent that detects susceptibility to prostate cancer gene, comprises PCR primer and the test kit that contains this primer.

For achieving the above object, the present invention is by the following technical solutions:

Detecting a nucleotide sequence for susceptibility to prostate cancer, is nucleotide sequence shown in sequence table SEQ ID No.1.Described nucleotide sequence is the nucleotide fragments that RFX6 gene the 11 intron comprises single thuja acid pleomorphism site rs1321366.Fig. 1 is the schematic diagram in RFX6 gene structure and rs1321366 polymorphic variation site, and RFX6 gene includes 17 introns, and rs1321366 site is marked on the corresponding position of the 11 intron in RFX6 gene map.

When the genotype of described single thuja acid pleomorphism site rs1321366 is GG, susceptibility to prostate cancer is the highest; When genotype is AG or AA, susceptibility to prostate cancer is lower.

One group is detected the primer of susceptibility to prostate cancer, can increase and obtain the nucleotide sequence of the detection susceptibility to prostate cancer that RFX6 gene the 11 intron comprises single thuja acid pleomorphism site rs1321366.

The nucleotide sequence of described primer is respectively shown in sequence table SEQ ID No.2 and sequence table SEQ ID No.3.

A detection method for susceptibility to prostate cancer gene, comprises the steps:

(1) genomic dna of extracting sample, the nucleotide fragments that comprises single thuja acid pleomorphism site rs1321366 of amplification RFX6 gene the 11 intron;

(2) genotype of single thuja acid pleomorphism site rs1321366 in detecting step (1) product, when genotype is GG, susceptibility to prostate cancer is the highest; When genotype is TG or TT, susceptibility to prostate cancer is lower.

Nucleotide fragments in described step (1) is the nucleotide sequence shown in sequence table SEQ ID No.1, rs1321366 site be positioned at this nucleotide sequence+501.

The nucleotide fragments that comprises single thuja acid pleomorphism site rs1321366 of described amplification RFX6 gene the 11 intron, the nucleotide sequence of one group of primer of use is respectively shown in sequence table SEQ ID No.2 and SEQ ID No.3.

The invention provides a kind of diagnostic kit that detects susceptibility to prostate cancer, the conventional assembly, reagent, damping fluid etc. of the test kit that wherein contains the primer pair in specific amplification RFX6 gene intron rs1321366 of the present invention site and detect for pcr amplification, those skilled in the art know these conventional assembly and detection methods.Whole components, content, source and using method in test kit of the present invention are as follows:

Predict a test kit of PCa, detect application for 10 person-portions, formed by following reagent:

10 μ L 10 × PCR damping fluids (purchased from Pharmacia),

2 μ L 10mM dNTP mixed solutions (purchased from Pharmacia),

2 μ L Taq archaeal dna polymerases (2unit/ μ L) (purchased from Takara),

1 μ L F1 primer, is the nucleotide sequence shown in SEQ ID NO.2, and concentration is 10pmol/ μ L;

1 μ L R1 primer, is the nucleotide sequence shown in SEQ ID NO.3, and concentration is 10pmol/ μ L;

8 μ L 10 × LC-green PLUS saturated fluorescence dyestuffs; (purchased from American I daho company)

The each 0.5 μ L of 2 μ L oligonucleotide internal reference primer, concentration is 10pmol/ μ L, wherein low temperature internal reference primers F is the nucleotide sequence shown in SEQ ID NO.4, low temperature internal reference primer R is the nucleotide sequence shown in SEQ ID NO.5, high temperature internal reference primers F is the sequence shown in SEQ ID NO.6, and high temperature internal reference primer R is the nucleotide sequence shown in SEQ ID NO.7;

64 μ L pure water.

Using method:

1) pcr amplification: by the intron Partial Fragment of pcr amplification RFX6 gene, prepare mixed solution: 10 × PCR reaction buffer, 1 μ L, 10mmol/L dNTP 0.2 μ L, TaqDNA polysaccharase 0.2 μ L, 10pmol/L upstream primer 0.1 μ L, 10pmol/L downstream primer 0.1 μ L, 10 × LC Green PLUS saturated fluorescence dyestuff, 0.8 μ L, the each 0.05 μ L of oligonucleotide internal reference 0.2 μ L(high and low temperature oligonucleotide internal reference upstream and downstream primer) (sequence is in table 1), genomic dna 1 μ L, adds deionized water to 10 μ L.PCR reaction conditions is 95 DEG C of denaturation 5min, 95 DEG C of sex change 1min, and 54 DEG C of annealing 30s, 72 DEG C are extended 6s, 45 circulations altogether, 72 DEG C of total elongation 7min.Before carrying out high-resolution fusion curve analysis, carry out denature and renature processing: 95 DEG C of 30s, 25 DEG C of 2min, 94 DEG C of 30s, 24 DEG C of 4min.When PCR, in each system, add the paraffin oil of 20 μ l, evaporate preventing.

2) genotype is judged: PCR product is moved in special 96 orifice plates of HRM, on Light scanner TMHR-I96, carry out HRM analysis, with Light Scanner Call IT software, the curve after gathering is analyzed, judged genotype according to the difference of melting curve.

The purposes of RFX6 gene the 11 intron list thuja acid pleomorphism site rs1321366 in reagent or the medicine of preparation diagnosis or treatment prostate cancer.

Measuring method of the present invention has been measured the genomic dna that derives from people, and sample does not limit, as: body fluid (blood, ascites and urine etc.), histocyte (as hepatic tissue) etc.Can prepare genomic dna by extraction and these samples of purifying.The concentration of adjusting genomic dna, makes it consistent as much as possible.Taking genomic dna as template, can amplify the nucleic acid fragment containing RFX6 intragenic mutation site rs1321366, to obtain the great amount of samples of mensuration.This sample that contains the DNA fragmentation acquisition of RFX6 intragenic mutation point by amplification, is particularly suitable for as measuring material.

In the time carrying out gene auxiliary diagnosis, the present invention is preferably applied in and measures the auxiliary diagnostic existing according to RFX6 intron rs1321366 mutation type, auxiliary diagnostic comprises the particular agent as neccessary composition, and it is corresponding to the method for measuring rs1321366 gene mutation type.Select suitable particular agent by the measuring method adopting, as DNA fragmentation and/or for the primer of pcr amplification step.

Advantage of the present invention is: the present invention has illustrated the dependency of RFX6 intron rs1321366 pleomorphism site and PCa first, a kind of method and test kit of the PCa of prediction susceptibility are provided, the method can be used for prevention, the auxiliary diagnosis of PCa, can also be used for new drug development.

Below in conjunction with the drawings and specific embodiments, the present invention is further described; so that the public has a better understanding summary of the invention; not limitation of the present invention, all any this areas of doing according to the disclosure of invention be equal to replacement, all belong to protection scope of the present invention.

Brief description of the drawings

Fig. 1 is the schematic diagram in RFX6 gene structure and intron polymorphic variation site

Fig. 2 is that RFX6 gene intron rs1321366 site is through HRM somatotype solubility curve figure

Fig. 3 is the sequencer map in RFX6 gene intron rs1321366 site

Embodiment

Represent that for the following example the english abbreviation of reagent is as follows:

10 × PCR damping fluid: 10mM Tris-HCI (pH=8.3), 500mM Repone K (KCI), 10mM magnesium chloride (MgCI2), 0.01% (W/V) gelatinum

DNTP: deoxynucleoside triphosphate

EDTA: disodium ethylene diamine tetraacetate

TE：10mM?Tris-HCI(pH=7.5)，1mM?EDTA(pH=8.0)

Embodiment 1: the extraction of blood sample collection and genomic dna:

(1) PCa patient is all through histopathologic diagnosis, choose altogether the PCa patient's 273 examples (age: 46 – 93 years old from Beijing and Efficiency in Buildings in Tianjin Area consanguinity-less relation, average 72.3 years old), with the contrast 606 examples (age: 58 – 94 years old of regional age-matched, average 70.4 years old), be the male sex, without PCa family history, DRE feminine gender and PSA<4ng/mL.All persons under inspection are Han nationality and sign written Informed Consent Form, and this research obtains Beijing Hospital, and the accreditation of ethics audit committee of Gerontological Research Center institute of the Ministry of Health, meets AMM's Declaration of Helsinki: the ethic principle of human medical research.(2) according to following method, preparation human gene group DNA.1. first in the 1.5mLEP pipe of label, add 1000 μ L erythrocyte cracked liquids, after add 400 μ LEDTA anticoagulations (anticoagulation is put upside down and mixed 3-5 time before adding), put upside down and mix, room temperature leaves standstill 10 minutes; 2. 13000rpm, after centrifugal 30 seconds, removes supernatant liquor; 3. in gained precipitation, add 480 μ l nucleic acid lysates, attack tube wall, adds 20 μ L Proteinase Ks (diluting 20 times of diluted protein enzyme K with splitting karyolymph) after fully mixing, and puts upside down and mixes, 65 DEG C of incubators 10 minutes, (frequently mix up and down during this time, guarantee without grumeleuse); 4. after taking out, be down to room temperature, add 300 μ L albumen precipitation liquid, fully put upside down and mix, leave standstill 10 minutes, centrifugal 2 minutes of 13000rpm; 5. supernatant liquor is moved in new EP pipe, add the Virahol of 670 μ L precoolings, fully put upside down and mix (more than 10 times), visible linear DNA forms little agglomerate, centrifugal 2 minutes of 13000rpm gradually; 6. abandon supernatant liquor and guarantee to precipitate and stay in EP, adding 670 μ L 70% ethanol, turning upside down and mix, centrifugal 2 minutes of 13000rpm; 7. abandon supernatant, make to manage interior ethanol volatilization clean; 8. add TE lysate (400 μ L), fully dissolve, then the genomic dna extracting is carried out to the analysis of concentration and purity, draw part DNA solution as working fluid, concentration correction is to 20ng/ μ L, be positioned over 4 DEG C for subsequent use, residue genomic dna is put-20 DEG C of Refrigerator stores.

The identification qualification of embodiment 2 SNP

The present invention adopt PCR-high resolving power solubility curve (HRM) analytical method and PCR sequencing technologies simultaneously the genotype of the rs1321366 site of the intron gene regions to RFX6 (its loci is A/G) detect.

1) determining of PCR-HRM primer

From Genebank, look near the DNA base sequence (SEQ ID NO.1) of getting rs1321366, design of primers completes under Oligo 6.0 and primer5.0 software.Object fragment is positioned at RFX6 gene intron district, and total length 63bp has determined positive-sense strand F1(+458bp--+475bp) and antisense strand R1(+504bp--+520bp), specific primer sequence is as follows:

F1：5’-CAGCTTTCCCACTAATGT-3’（SEQ?ID?NO.2）

R1：5’-AAATGCAACGTGAGACC-3’（SEQ?ID?NO.3）

2) PCR-HRM reaction system and condition

Place, the rs1321366 site fragment of the intron gene regions by pcr amplification RFX6, PCR reaction system is: 10 × PCR reaction buffer, 1 μ L, 10mmol/L dNTP 0.2 μ L, Taq archaeal dna polymerase 0.2 μ L, 10pmol/L upstream primer 0.1 μ L, 10pmol/L downstream primer 0.1 μ L, 10 × LC Green PLUS saturated fluorescence dyestuff, 0.8 μ L, 0.2 μ L(is high for oligonucleotide internal reference, on low temperature oligonucleotide internal reference, the each 0.05 μ L of downstream primer), 3 ' end C3 sealing, stop and extend (in table 1), genomic dna 1 μ L, add deionized water to 10 μ L.The paraffin oil that adds 20 μ l when PCR in each system, prevents from evaporating.PCR reaction conditions is 95 DEG C of denaturation 5min, 95 DEG C of sex change 1min, and 54 DEG C of annealing 30s, 72 DEG C are extended 6s, 45 circulations altogether, 72 DEG C of total elongation 7min.Before carrying out high-resolution fusion curve analysis, carry out denature and renature processing: 95 DEG C of 30s, 25 DEG C of 2min, 94 DEG C of 30s, 24 DEG C of 4min.

Table 1 high and low temperature oligonucleotide internal reference primer sequence, annealing temperature and product sheet segment length

3) HRM judges genotype

PCR product is moved in special 96 orifice plates of HRM, on Light scanner TMHR-I96, carry out HRM analysis: since 45 DEG C, slope with 0.3 DEG C/s gathers melting curve, to 98 DEG C of end, with Light Scanner Call IT software, the curve (Fig. 2) after gathering is analyzed, judged genotype.

4) sequence verification

From the individuality of the different genotype of gained, randomly draw respectively 3 routine samples and carry out sequence verification.Order-checking sample re-starts pcr amplification, and sequencing primer sequence is: F2:5 '-CAGCTTTCCCACTAATGT-3 ' (SEQ ID No.8), R2:5 '-ATCTTGCCCAGGTATGTT-3 ' (SEQ ID No.9), the long 188bp of amplified fragments.PCR reaction is totally 30 μ L, comprises: genomic dna 3 μ L, 10 × PCR Buffer, 3 μ L, 10mmol/LdNTP 0.6 μ L, Taq archaeal dna polymerase (5U/ μ L) 0.6 μ L, the each 0.3 μ L of upstream and downstream primer (10pmol/ μ L), deionized water is supplemented to cumulative volume 30 μ L.PCR reaction conditions is: 95 DEG C of denaturation 5min laggard enter major cycle, 95 DEG C of sex change 30s, 60 DEG C of annealing 30s, 72 DEG C are extended 60s, 35 circulations, 72 DEG C are extended 7min.PCR product detects through 8% polyacrylamide gel electrophoresis, after gel imaging system observation is qualified, send Hua Da gene sequencing portion to carry out sequence verification (Fig. 3).

The dependency of embodiment 3 gene SNPs and PCa

Statistical method: colony's representativeness of using Hardy-Weinberg balance check research sample.Utilize SPSS11.0 software Pearson chi square test to calculate allelotrope, the distribution frequency of genotype between PCa case group and Normal group of RFX6 gene intron rs1321366 pleomorphism site, logistic returns ill risk OR value and the 95%CI credibility interval thereof of evaluating PCa, taking P<0.05 as significance of difference standard.

Result: be positioned at the distribution between case and control group of the genotype of SNP rs1321366 polymorphic site of RFX6 gene intron and gene frequency and refer to table 2 with the association analysis of PCa.

The genotype of table 2 RFX6 gene intron SNP rs1321366 pleomorphism site and gene frequency in Chinese population the distribution between case-control group and with the association analysis of PCa

Note: OR: odds ratio; CI: credibility interval.HWE:Hardy-Weinberg balance.

From table 2, the G allelotrope in the rs1321366 site of RFX6 gene intron, on its DNA complementary strand, be C allelotrope, distribution frequency in patient colony is significantly higher than its distribution frequency (70.0%vs.62.1%) in healthy normal population, there is significant difference (ageadjustment P=0.003), and the OR value in G site is 1.39,95%CI:1.12-1.73; In recessive model (G/G vs.A/A+A/G), the distribution frequency of its genotype in case group is significantly higher than (the ageadjustment P=0.001) in control group, and allelotrope and genotype and PCa association analysis all show that the G allelotrope in the rs1321366 site of RFX6 gene intron may

Be proportionate with PCa is ill, likely increase the onset risk of PCa.

Embodiment 4 detection kit

Preparation detects the test kit of PCa relevant risk, includes the primer pair in the rs1321366 site that can amplify RFX6 gene intron, and other PCR-HRM corresponding reagent.Test kit of the present invention detects application for 10 person-portions, keeps in Dark Place in-20 DEG C, and its component, content and source comprise:

10 μ L 10 × PCR damping fluids (Pharmacia),

2 μ L 10mM dNTP mixed solutions (Pharmacia),

2 μ L Taq archaeal dna polymerases (2unit/ μ L) (Takara),

1μL?F1（SEQ?ID?NO.2）（10pmol/μL），

1 μ L R1(SEQ ID NO.3) (10pmol/ μ L) primer, (the raw work in Shanghai is synthetic)

8 μ L 10 × LC-green PLUS saturated fluorescence dyestuffs (American I daho company),

2 μ L oligonucleotide internal references (10pmol/ μ L) (the each 0.5 μ L of high and low temperature oligonucleotide internal reference upstream and downstream primer), (sequence is in table 1),

64 μ L pure water.

Proof test: adopt this test kit, choose at random PCa clinical samples 10 examples, control group sample 10 examples, through the polymorphism in PCR-HRM detection RFX6 gene the 11 intron rs1321366 site.

One. method

1.PCR amplification: by the intron Partial Fragment of pcr amplification RFX6 gene, prepare mixed solution: 10 × PCR reaction buffer, 1 μ L, 10mmol/L dNTP 0.2 μ L, TaqDNA polysaccharase 0.2 μ L, 10pmol/L upstream primer 0.1 μ L, 10pmol/L downstream primer 0.1 μ L, 10 × LC Green PLUS saturated fluorescence dyestuff, 0.8 μ L, the each 0.05 μ L of oligonucleotide internal reference 0.2 μ L(high and low temperature oligonucleotide internal reference upstream and downstream primer) (sequence is in table 1), genomic dna 1 μ L, adds deionized water to 10 μ L.PCR reaction conditions is 95 DEG C of denaturation 5min, 95 DEG C of sex change 1min, and 54 DEG C of annealing 30s, 72 DEG C are extended 6s, 45 circulations altogether, 72 DEG C of total elongation 7min.Before carrying out high-resolution fusion curve analysis, carry out denature and renature processing: 95 DEG C of 30s, 25 DEG C of 2 min, 94 DEG C of 30s, 24 DEG C of 4min.When PCR, in each system, add the paraffin oil of 20 μ l, evaporate preventing.

2. genotype is judged: PCR product is moved in special 96 orifice plates of HRM, on Light scanner TMHR-I96, carry out HRM analysis, with Light Scanner Call IT software, the curve after gathering is analyzed, judged genotype according to the difference of melting curve.

Two. result:

Result demonstration, the genotype in PCa clinical samples RFX6 gene the 11 intron rs1321366 site is GG; The genotype in control group sample rs1321366 site is AG or AA.

The present invention has the illustration of practicality:

The detection method of the rs1321366 polymorphism of RFX6 gene intron of the present invention can be used for the G loci in this site on analyst's genomic dna, on its DNA complementary strand, be C loci, be applied to the complementary diagnosis to PCa, can assess individuality has the ill risk of great PCa, is beneficial to carry out early prevention and the treatment of PCa.

Utilize the present invention to set forth the nucleotide variation in the rs1321366 site of RFX6 gene intron, as one of biomarker, can be used as the screening of the molecular target of medicinal design, there is to help to find the bioactive molecule that regulates RFX6 genetic expression, promote PCa new drug development.

The nucleotide sequence of rs1321366 loci polymorphism of the detection RFX6 gene intron that the present invention sets up, can highly sensitive, the specific test kit that is applied to PCa gene auxiliary diagnosis.

As above tell, reach a conclusion, the polymorphism in the rs1321366 site of RFX6 gene intron and PCa tool significant correlation.Therefore, measure this polymorphism according to the present invention, can be used for the gene auxiliary diagnosis of PCa.

The present invention has narrated the relevant new mutant site of RFX6 gene intron PCa, and a kind of method of measuring gene pleiomorphism is provided, and, according to the present invention, only need a small amount of DNA sample to be just enough to measure the rs1321366 polymorphism of RFX6 gene intron.

The invention provides a kind of gene aided diagnosis method of the PCa of mensuration related gene polymorphism.

Claims (1)

1. detect a test kit for prostate cancer tumor susceptibility gene, it is characterized in that being formed by following reagent:

10 μ L10 × PCR damping fluids;

2 μ L10mM dNTP mixed solutions;

2 μ L Taq archaeal dna polymerases, 2unit/ μ L;

1 μ L F1 primer, is the nucleotide sequence shown in SEQ ID No.2, and concentration is 10pmol/ μ L;

1 μ L R1 primer, is the nucleotide sequence shown in SEQ ID No.3, and concentration is 10pmol/ μ L;

8 μ L10 × LC-green PLUS saturated fluorescence dyestuffs;

The each 0.5 μ L of 2 μ L oligonucleotide internal reference primer, concentration is 10pmol/ μ L, wherein low temperature internal reference primers F is the nucleotide sequence shown in SEQ ID NO.4, low temperature internal reference primer R is the nucleotide sequence shown in SEQ ID NO.5, high temperature internal reference primers F is the sequence shown in SEQ ID NO.6, and high temperature internal reference primer R is the nucleotide sequence shown in SEQ ID NO.7;

64 μ L pure water.