CN103205422B - Method and detection kit for prediction of prostate cancer susceptibility - Google Patents

Abstract

The invention discloses a method and a detection kit for prediction of prostate cancer susceptibility. According to the invention, genomic DNA of a host cell is extracted and the genotype of site rs9400968 on a fourth intron of the RFX6 gene of a subject is determined so as to predicate susceptibility of the subject to a prostate cancer; if the genotype of the rs9400968 on the intron of the RFX6 gene is GG, the subject has highest susceptibility, and if the genotype of the rs9400968 is AG or AA, the subject has low susceptibility. The invention has the following advantages: correlation between the rs9400968 gene polymorphism site and the prostate cancer is elaborated for the first time; the method for predicting prostate cancer susceptibility is provided; and the method is applicable to early prevention and diagnosis and auxiliary diagnosis of the prostate cancer and can also be used for research and development of a novel drug.

Description

A kind of method and detection kit predicting susceptibility to prostate cancer

Technical field

The present invention relates to a kind of method and the detection kit of predicting susceptibility to prostate cancer, by measuring with relating to prostate cancers because rs9400968 loci polymorphism on the intron of RFX6 predicts the susceptibility of experimenter for prostate cancer in particular, 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) occurs in modal malignant tumour in male reproductive system.The U.S., after adding up 1975-2006 cancer morbidity, estimates that PCa new cases in 2010 are 217,730 examples, and occupy the male sex and to swell well in knurl first, the death toll of PCa is estimated as 32,050, in male tumor mortality ratio, occupy second.Age is the important risk factor of PCa, and the basic seldom morbidity at less than 45 years old age, increases with the growth at age.American Cancer Society adds up, the male sex PCa sickness rate of less than 40 years old be 1/8499,40-59 year the male sex in be be then 1/8 in more than 1/15, the 70 years old male sex in 1/38,60-69 years old male sex.Along with our countries population's aging, the sickness rate of PCa rises just year by year in China, Shanghai Standardized incidence rate rises to 5.5/10 ten thousand in 1997 ~ 1999 years from 1.8/10 ten thousand in 1973 ~ 1975 years, the sickness rate of part counties and cities, southern areas, Guizhou Province PCa has risen to 4.28/10 ten thousand in 2004 ~ 2008 years from 1.72/10 ten thousand in 1994 ~ 1998 years, become the common problem threatening China's men's health.

Carry out the research of PCa inherited pathogenic factor at present, the extensive genome-wide association study of many employings (Genome-WideAssociation Studies, GWAS) checking and in the many crowds of small sample, and multiple PCa susceptible SNPs of GWAS qualification have been replicated and have determined in multiple different crowd, demonstrate the association analysis method validity in PCa genetic research of SNPs as genomic marker.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, 70.1% is had for the conversion between homotype base: as G/A or T/C, 29.1% for occurring in the transversion between purine and pyrimidine in this single base change.SNPs contains the 80-90% of 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 with its density high (average every 1kb just have 1), representative strong (SNPs being arranged in gene internal 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 for biallelic marker, can 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, there are B, C and D tri-structural domains, mainly express at pancreas, lower than expression level with family RFX with other, be crucial transcriptional in the development of pancreas and the adjustment of function.RFX6 and RFX2 and RFX3 direct effect, and the latter two are expressed at pancreas and play a role.

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 cell differentiation.The sudden change of these two kinds of genes shows similar and different phenotype.Mankind RFX6 suddenlys change, and can cause intractable diarrhoea and diabetes, intestinal occlusion and anomalies of biliary system, neonatal diabetes.Contribute to illustrating the formation of pancreas islet and β cell, the pathogenic process of diabetes to fully understanding of Rfx6.

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

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

2010, Takata R etc. identified rs339331 and the PCa risk association be 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 association section of an about 200kb, this region is contained two gene: GPRC6A, RFX6 combines the above analysis to RFX6 function, we infer that RFX6 gene is PCa tumor susceptibility gene and have selected the rs9400968 being positioned at it, in 273 PCa patients and 606 contrast crowds, carry out association analysis after somatotype, result determines rs9400968 and associates with Chinese population PCa.

Rs9400968 on chromosome position is 117,317,298, be positioned on the 4th intron of RFX6, this intron has 84598bp, rs9400968 is the SNP site of distance initiating terminal 50669, 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 rs9400968 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 predicting susceptibility to prostate cancer.

Second object of the present invention is to provide a kind of reagent predicting susceptibility to prostate cancer, comprises PCR primer and the test kit containing 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 nucleotides sequence is classified as nucleotide fragments RFX6 gene intron comprising single nucleotide polymorphism site rs9400968.Namely rs9400968 site is the+501 bit base R (R=A or G).Fig. 1 is the schematic diagram in RFX6 gene structure and rs9400968 polymorphic variation site, and RFX6 gene includes 17 introns, and rs9400968 site is marked on the 4th intron corresponding position in RFX6 gene map.

The present invention studies discovery, and when the genotype of described single nucleotide polymorphism site rs9400968 is GG, susceptibility to prostate cancer is the highest; When genotype is AG or AA, susceptibility to prostate cancer is lower.

1. one group is detected the primer of susceptibility to prostate cancer, can increase and obtain RFX6 gene intron comprises the nucleotide fragments of single nucleotide polymorphism site rs9400968.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 comprising single nucleotide polymorphism site rs9400968 of amplification RFX6 gene and GPRC6A gene interval;

(2) genotype of single nucleotide polymorphism site rs9400968 in detecting step (1) product, when genotype is GG, susceptibility to prostate cancer is the highest; When genotype is AG or AA, susceptibility to prostate cancer is lower.

Nucleotide fragments in described step (1) is the nucleotide sequence shown in sequence table SEQ ID No.1, and rs9400968 site is positioned at+501 of this nucleotide sequence.

Described amplification RFX6 gene intron comprises the nucleotide fragments of single nucleotide polymorphism site rs9400968, 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 detecting susceptibility to prostate cancer, the general components, reagent, damping fluid etc. of the primer pair wherein containing specific amplification RFX6 gene intron rs9400968 site of the present invention and the test kit for pcr amplification detection, those skilled in the art know these general components and detection method.Whole components in test kit of the present invention, content, source and using method are as follows:

Predict a test kit of PCa, detect application for 10 person-portions, be made up of following reagent:

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

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

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

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

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

8uL 10 × LC-green PLUS saturated fluorescence dyestuff; (purchased from American Idaho company)

The each 0.5uL of 2uL 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 SEQID NO.7;

64uL 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, 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, oligonucleotide internal reference 0.2 μ L (each 0.05 μ L of 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 extend 6s, altogether 45 circulations, 72 DEG C of total elongation 7min.Before carrying out high-resolution fusion curve analysis, carry out denature and renature process: 95 DEG C of 30s, 25 DEG C of 2min, 94 DEG C of 30s, 24 DEG C of 4min.In each system, the paraffin oil of 20 μ l is added, to prevent from evaporating during PCR.

2) genotype judges: PCR primer moved in special 96 orifice plates of HRM, Light scanner TMHR-I96 carries out HRM analysis, analyze the curve after collection with Light Scanner Call IT software, the difference according to melting curve judges genotype.

Nucleotide sequence shown in sequence table SEQ ID No.1 and the nucleotide fragments comprising single nucleotide polymorphism site rs9400968 thereof are preparing the purposes in the reagent or medicine of diagnosing or treating prostate cancer.

The purposes of single nucleotide polymorphism site rs9400968 in the reagent preparing diagnosis or treatment prostate cancer or medicine on RFX6 gene intron.

Measuring method of the present invention determines the genomic dna deriving from people, and sample does not limit, as: body fluid (blood, ascites and urine etc.), histocyte (as hepatic tissue) etc.Genomic dna can be prepared by these samples of Isolation and purification.The concentration of adjustment genomic dna, makes it consistent as much as possible.Take genomic dna as template, the nucleic acid fragment containing RFX6 intragenic mutation site rs9400968 can be amplified, to obtain the great amount of samples of mensuration.This sample obtained containing the DNA fragmentation of RFX6 intragenic mutation point by amplification, is particularly suitable for as mensuration material.

When carrying out gene auxiliary diagnosis, the present invention is preferably applied in the auxiliary diagnostic measuring and exist according to RFX6 intron rs9400968 mutation type, auxiliary diagnostic comprises the particular agent as neccessary composition, and it corresponds to the method for measuring rs9400968 gene mutation type.Suitable particular agent is selected, as DNA fragmentation and/or the primer for pcr amplification step by the measuring method adopted.

Advantage of the present invention is: the present invention illustrates the dependency of RFX6 intron rs9400968 pleomorphism site and PCa first, provides a kind of method predicting PCa susceptibility, and 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, the equivalent replacement of all any this areas of doing according to the disclosure of invention, all belongs to protection scope of the present invention.

Accompanying drawing explanation

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

Fig. 2 is that RFX6 gene intron rs9400968 site is through HRM somatotype solubility curve figure

Fig. 3 is the sequencer map in RFX6 gene intron rs9400968 site

Embodiment

As follows for representing the english abbreviation of reagent in the following example:

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-HCl(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 the PCa patient 273 example (age: 46-93 year from Beijing and Efficiency in Buildings in Tianjin Area consanguinity-less relation altogether, average 72.3 years old), with area age-matched contrast 606 example (age: 58-94 year, average 70.4 years old), be the male sex, the negative and PSA < 4ng/mL without PCa family history, DRE.All persons under inspection are Han nationality and signature Written informed consent, 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 1000uL erythrocyte cracked liquid, after add 400 μ LEDTA anticoagulations (anticoagulation puts upside down mixing 3-5 time before adding), put upside down mixing, standing 10 minutes of room temperature; 2. 13000rpm is after centrifugal 30 seconds, removing supernatant liquor; 3. in gained precipitation, adding 480 μ l nucleic acid cleavage liquid, attack tube wall, fully adding 20 μ L Proteinase Ks (diluting 20 times of diluted protein enzyme K with splitting karyolymph) after mixing, put upside down mixing, 65 DEG C of incubators 10 minutes, (period mixes frequently up and down, guarantees without grumeleuse); 4. be down to room temperature after taking out, add 300 μ L albumen precipitation liquid, fully put upside down mixing, leave standstill 10 minutes, centrifugal 2 minutes of 13000rpm; 5. moved to by supernatant liquor in new EP pipe, add the Virahol of 670uL precooling, fully put upside down mixing (more than 10 times), visible linear DNA forms little agglomerate gradually, centrifugal 2 minutes of 13000rpm; 6. abandon supernatant liquor and guarantee that precipitation is stayed in EP, adding 670uL 70% ethanol, mixing of turning upside down, centrifugal 2 minutes of 13000rpm; 7. abandon supernatant, make ethanol volatilization in pipe clean; 8. add TE lysate (400uL), fully dissolve, then the genomic dna extracted is carried out to the analysis of concentration and purity, draw part DNA solution as working fluid, concentration correction to 20ng/ μ L, be positioned over 4 DEG C for subsequent use, residue genomic dna put-20 DEG C of Refrigerator stores.

The identification qualification of embodiment 2:SNP

The present invention adopts PCR-high resolving power solubility curve (HRM) analytical method and PCR sequencing technologies to detect the genotype in the rs9400968 site (its loci is A/G) of the intron gene regions of RFX6 simultaneously.

The determination of 1.PCR-HRM primer

From Genebank, look into the DNA base sequence (SEQ ID NO.1) got near rs9400968, design of primers completes under Oligo 6.0 and primer5.0 software.Object fragment is positioned at RFX6 gene intron district, total length 86bp, and determine positive-sense strand F1 (+439bp--+457bp) and antisense strand R1 (+504bp--+524bp), specific primer sequence is as follows:

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

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

2.PCR-HRM reaction system and condition

By place, the rs9400968 site fragment of the intron gene regions of 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 GreenPLUS saturated fluorescence dyestuff 0.8 μ L, oligonucleotide internal reference 0.2 μ L is (high, on low temperature oligonucleotide internal reference, the each 0.05 μ L of downstream primer) 3 ' end C3 close, stop and extend, in table 1), genomic dna 1 μ L, add deionized water to 10 μ L.In each system, add the paraffin oil of 20 μ l during PCR, prevent 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 extend 6s, altogether 45 circulations, 72 DEG C of total elongation 7min.Before carrying out high-resolution fusion curve analysis, carry out denature and renature process: 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 primer is moved in special 96 orifice plates of HRM, Light scanner TMHR-I 96 carries out HRM analysis: from 45 DEG C, melting curve is gathered with the slope of 0.3 DEG C/s, to 98 DEG C of end, with LightScanner Call IT software, the curve (Fig. 2) after collection is analyzed, judge genotype.

4. sequence verification

From the individuality of the different genotype of gained, randomly draw 3 routine samples respectively carry out sequence verification.Order-checking sample re-starts pcr amplification, sequencing primer sequence is: F2:5 '-TGTCAGAAGGTGGTTATTG-3 ' (SEQ ID No.8), R2:5 '-CATAAATAGCTTGCCATAGC-3 ' (SEQ ID No.9), the long 427bp of amplified fragments.PCR reaction is totally 30 μ L, comprise: genomic dna 3 μ L, 10 × PCRBuffer 3 μ L, 10mmol/L dNTP 0.6 μ L, Taq archaeal dna polymerase (5U/ μ L) 0.6 μ L, upstream and downstream primer (10pmol/ μ L) each 0.3 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 extend 60s, 35 circulations, 72 DEG C of extension 7min.PCR primer detects through 8% polyacrylamide gel electrophoresis, send Hua Da gene sequencing portion to carry out sequence verification (Fig. 3) after gel imaging system observation is qualified.

Embodiment 3: the dependency of gene SNP and PCa

Statistical method: the populational representation using Hardy-Weinberg balance check research sample.SPSS11.0 software Pearson chi square test is utilized to calculate allelotrope, the distribution frequency of genotype between PCa case group and Normal group of RFX6 gene intron rs9400968 pleomorphism site, logistic returns the risk OR value and 95%CI credibility interval thereof of evaluating PCa, is significance of difference standard with P < 0.05.

Result: be positioned at the distribution between case and control group of the genotype of the SNP rs9400968 polymorphic site of RFX6 gene intron and gene frequency and refer to table 2 with the association analysis of PCa.

The genotype of table 2RFX6 gene intron SNP rs9400968 pleomorphism site and the distribution of gene frequency in Chinese population between case-control group and the association analysis with PCa

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

From table 2, the G allelotrope in the rs9400968 site of RFX6 gene intron, namely be C allelotrope on its DNA complementary strand, distribution frequency in PATIENT POPULATION is significantly higher than its distribution frequency (63.6%vs.558%) in healthy normal population, there is significant difference (ageadjustment P=0.003), and the OR value in G site is 1.38,95%CI:1.11-1.70; In recessive model (G/G vs.A/A+A/G), the distribution frequency of its genotype in case group is significantly higher than (ageadjustment P=3.4 × 10 in control group ^-4), allelotrope and genotype and PCa association analysis all show that the G allelotrope in the rs9400968 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 rs9400968 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, and keep in Dark Place in-20 DEG C, its component, content and source comprise:

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

2 μ L 10mM dNTP mixed solution (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 synthesis in Shanghai)

8uL 10 × LC-green PLUS saturated fluorescence dyestuff (American I daho company),

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

64uL pure water.

Proof test: adopt this test kit, random selecting PCa clinical samples 10 example, control group sample 10 example, detects the polymorphism in the rs940096g site of RFX6 gene intron through PCR-HRM.

1. method:

1.PCR increases: by the Partial Fragment of pcr amplification RFX6 gene regions, prepare mixed solution: 10 × PCR reaction buffer 1 μ L, 10mmol/L dNTP 0.2 μ L, Taq 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, oligonucleotide internal reference 0.2 μ L (each 0.05 μ L of 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 extend 6s, altogether 45 circulations, 72 DEG C of total elongation 7min.Before carrying out high-resolution fusion curve analysis, carry out denature and renature process: 95 DEG C of 30s, 25 DEG C of 2min, 94 DEG C of 30s, 24 DEG C of 4min.In each system, the paraffin oil of 20 μ l is added, to prevent from evaporating during PCR.

2. genotype judges: PCR primer moved in special 96 orifice plates of HRM, Light scannerTMHR-I 96 carries out HRM analysis, analyze the curve after collection with Light Scanner Call IT software, the difference according to melting curve judges genotype.

Two. result:

Result shows, and the genotype in PCa clinical samples rs9400968 site is GG; The genotype in control group sample rs9400968 site is AG or AA.

The present invention has the illustration of practicality:

The detection method of the rs9400968 polymorphism of RFX6 gene intron of the present invention can be used for the G loci in this site on analyst's genomic dna, namely be C loci on its DNA complementary strand, be applied to the complementary diagnosis to PCa, individuality can be assessed and have great PCa risk, be beneficial to early prevention and the treatment of carrying out PCa.

The present invention is utilized to set forth the nucleotide variation in the rs9400968 site of RFX6 gene intron, as one of biomarker, can be used as the screening of the molecular target of medicinal design, to help to find the bioactive molecule having and regulate RFX6 genetic expression, promote PCa new drug development.

The nucleotide sequence of rs9400968 loci polymorphism of the detection RFX6 gene intron that the present invention sets up, can highly sensitive, the specific test kit being applied to PCa gene auxiliary diagnosis.

As above told, reached a conclusion, the polymorphism in the rs9400968 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.

Invention describes the new mutant site that RFX6 gene intron PCa is relevant, and provide a kind of method measuring gene pleiomorphism, and, according to the present invention, only need a small amount of DNA sample to be just enough to measure the rs9400968 polymorphism of RFX6 gene intron.

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

Claims (2)

1. one group is detected the primer of susceptibility to prostate cancer, it is characterized in that: can increase and obtain RFX6 gene intron comprises the nucleotide fragments of single nucleotide polymorphism site rs9400968; The nucleotide sequence of described primer is respectively shown in sequence table SEQ ID No.2 and sequence table SEQ ID No.3.

2. detect a test kit for prostate cancer tumor susceptibility gene, it is characterized in that being made up of following reagent:

10 μ L 10 × PCR damping fluids;

2 μ L 10mM dNTP mixed solutions;

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

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

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

8 μ L 10 × 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 SEQID NO.7;

64 μ L pure water.