CN107058486A - Detect the primer sets and kit of nasopharyngeal carcinoma glycolysis related gene parting - Google Patents

Detect the primer sets and kit of nasopharyngeal carcinoma glycolysis related gene parting Download PDF

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CN107058486A
CN107058486A CN201611240834.5A CN201611240834A CN107058486A CN 107058486 A CN107058486 A CN 107058486A CN 201611240834 A CN201611240834 A CN 201611240834A CN 107058486 A CN107058486 A CN 107058486A
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王晖
赵宇
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Hunan Cancer Hospital
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Abstract

The present invention relates to a kind of primer sets for detecting nasopharyngeal carcinoma glycolysis related gene parting.The primer sets include the positive amplimers of GCK rs2244164 and reverse amplimer, the positive amplimers of HK2 rs656489 and reverse amplimer and the positive amplimers of HIF1 α rs10136168 and reverse amplimer.The invention further relates to a kind of kit for detecting nasopharyngeal carcinoma glycolysis related gene parting.The kit includes containing amplimer, PCR Buffer, MgCl2, the PCR premixed liquids of dNTP and Taq enzyme.The detection method of the present invention carries out Genotyping detection by combining multiple PCR technique, Single base extension technology and matrix solid-dispersion flying time mass spectrum analysis technology, with testing result is accurate, specificity is high, detection cycle is short, the sensitive advantage of detection method.

Description

Detect the primer sets and kit of nasopharyngeal carcinoma glycolysis related gene parting
Technical field
The present invention relates to beyond body nucleic acid detection technique field, more particularly to a kind of detection nasopharyngeal carcinoma glycolysis related gene point The primer sets and kit of type.
Background technology
Nasopharyngeal carcinoma (Nasopharyngeal carcinoma, NPC) is a kind of Highly invasive for coming from nasopharyngeal epithelium Malignant tumour.According to statistics, in 2012, the people of nasopharyngeal carcinoma new cases 86,700, the people of death 50,800, therefore it is considered as It is malignant tumour rarer in global range.But in epidemic regions, including southern china and Southeast Asia, the incidence of disease of nasopharyngeal carcinoma It can reach 15-50/100000.The region of anatomy of nasopharynx is deeper, around closes on many important blood vessels and nerve, special dissection The biological behaviour of position and local challenge growth causes operative treatment to be difficult to；And the histological type pair of most of nasopharyngeal carcinoma Radioactive ray are more sensitive, and current nasopharyngeal carcinoma generally acknowledges that effective treatment means are radiotherapy.The Nasopharyngeal Carcinoma Patients of early stage are using simple Radiotherapy has ideal effect, but because nasopharyngeal carcinoma early symptom is not obvious, during most of patient has been when medical In late period, tumor tissues are to around invading, or even there occurs DISTANT METASTASES IN, therefore radiation alone treats the overall survival of nasopharyngeal carcinoma It is unsatisfactory.Advanced Nasopharyngeal Carcinoma patient causes overall survival substantially to drop due to higher local relapse and Distant metastasis rates It is low, led to improve patients overall survival, chemotherapy is applied among the complex treatment of nasopharyngeal carcinoma.Although current NPC treatments take Pleasurable progress was obtained, but because nasopharyngeal carcinoma early symptom is not obvious and is easy to the growth side of local challenge and DISTANT METASTASES IN Formula so that be in middle and advanced stage during many patient assessments, such patient outcomes are obvious compared with early stage patient gap.
Therefore, the molecular marker for being effectively used for NPC clinical diagnosis and treatments is found, is early controlled for patient's early diagnosis, existence is improved Time is significant.
Tumour cell recodes metabolism to promote propagation to grow and long term survival.The metabolic process of this recodification it is most main It is characterized in that increased glucose uptake and glucose fermentation produce lactic acid, even in there is enough oxygen and normal mitochondria Can also occur in the case of function.This is referred to as Warburg effects.This effect is likely to be to be developed to malignant tumour early stage During indirect anoxic adaptation.Enhanced glycolysis can lose a large amount of production capacities of mitochondria, but while the change of interior environment Also promote tumour cell much more resistant to acid poisoning, provide the raw material and protection cell of biosynthesis macromolecular from oxidative stress Infringement.
SNP (Single nucleotide polymorphism, SNP) is that have necessarily in crowd Frequency (>1%) variation, specifically, be in genome due to single nucleotide variation (including insertion, replace, missing) make Changed into DNA sequence dna, so that human genome has more diversity.Many studies have shown that, all polygenes SNP and the generation development of a variety of diseases including tumour, prognosis and treatment susceptibility it is closely related, this prompting SNP It is potential to be applied to clinical prevention, examination, diagnosis and the guiding treatment for carrying out relevant disease, this for individualized treatment and Precisely treatment has very important significance.
At present, existing many research shows that glycolysis pathway gene SNP and kinds of tumors generation development are closely related, but sugar The change of glycolysis pathway gene is related to nasopharyngeal carcinoma risk to be had not been studied so far.Based on this, with the glycolysis path of nasopharyngeal carcinoma Key gene is point of penetration, to glycolytic pathway Hexokinase 2 (Hexokinase2, HK2), hypoxia-inducible factor lα (Hypoxia Inducible Factor-1 α, HIF-1 α), glucokinase (Glucokinase, GCK), lactic dehydrogenase 30 Tag SNPs of (Lactate dehydrogenase, LDH) 4 key genes are detected, this nasopharynx new to setting up Cancer Risk Screening technology is significant, can early stage, sensitive and stable screening for nasopharyngeal cancer risk, to carry out and When, effective monitoring and evaluation and intervention, reduce medical treatment cost, save social resources.
At present, the diagnosis in hospital for nasopharyngeal carcinoma is mainly also to rely on traditional pathology conventional method, examines in early days Disconnected rate is relatively low and preventive effect of for nasopharyngeal carcinoma is poor；In addition, not yet have for described glycolysis related gene GCK, HK2 and HIF1- α sequencing primer and corresponding detection kit emerges, therefore, appearance of the invention, for the body of nasopharyngeal carcinoma risk examination The foundation of outer diagnosis new method has great importance.
The content of the invention
In order to solve the diagnostic method of conventional nasopharyngeal carcinoma exist early diagnostic rate it is low and cause conditions of patients development deteriorate Technical problem, a kind of sensitiveness height of present invention offer, high specificity, detection cycle are short, the sugared ferment of the sensitive nasopharyngeal carcinoma of detection method The primer sets and kit of decorrelation Genotyping.
The present invention provides a kind of primer sets for detecting nasopharyngeal carcinoma glycolysis related gene parting, the glycolysis related gene Respectively GCK genes, HK2 genes and HIF1- α genes, the GCK genes, HK2 genes and HIF1- α gene polynorphisms site Respectively GCKrs2244164, HK2rs656489 and HIF1- α rs10136168, the primer sets include：
(1) for GCK rs2244164 sites,
Amplimer is：
GCK rs2244164 forward direction amplimers：
5’-ACGTTGGATGCATAATGTGTTTTCTCCGCC-3’(SEQ ID NO.1)；
The reverse amplimers of GCK rs2244164：
5’-ACGTTGGATGGGGACTTACTCCTGATCTAC-3’(SEQ ID NO.2)；
(2) for HK2rs656489 sites,
Amplimer is：
HK2rs656489 forward direction amplimers：
5’-ACGTTGGATGCTGGTGTCTAGCATTAGCTG-3’(SEQ ID NO.3)；
The reverse amplimers of HK2rs656489：
5’-ACGTTGGATGTGATCTTGGACATGGGATGG-3’(SEQ ID NO.4)；
(3) for HIF1- α rs10136168 sites,
Amplimer is：
HIF1- α rs10136168 forward direction amplimers：
5’-ACGTTGGATGTCACCCTCTGATGCAGTTAC-3’(SEQ ID NO.5)；
The reverse amplimers of HIF1- α rs10136168：
5’-ACGTTGGATGCAGCTCTGTTCTTTACTGCC-3’(SEQ ID NO.6)。
The present invention also provides a kind of kit for detecting nasopharyngeal carcinoma glycolysis related gene parting, and the kit includes containing There are the positive amplimers of above-mentioned GCK rs2244164 and the positive amplimer of reverse amplimer, HK2rs656489 and reverse The positive amplimer of amplimer, HIF1- α rs10136168 and reverse amplimer, PCR Buffer, MgCl2, dNTP and The PCR premixed liquids of Taq enzyme.
In a preferred embodiment of the kit that the present invention is provided, each reagent component is matched somebody with somebody in the PCR premixed liquids Than for：
In a preferred embodiment of the kit that the present invention is provided, the kit also includes：Positive reference substance And negative controls, the positive reference substance is saltant type standard items DNA, and the negative controls are wild type standard items DNA； Wherein described saltant type standard items DNA is respectively GCK rs2244164 mutant homozygous types and mutation heterozygous DNA (gene orders Referring to NCBI dbSNP database NC_000007.14), HK2rs656489 mutant homozygous type and mutation heterozygous DNA (gene sequences Row are referring to NCBI dbSNP database NC_000002.12), HIF1- α rs10136168 mutant homozygous types and mutation heterozygous DNA (gene order is referring to NCBI dbSNP database NC_000014.9)；Wherein described wild type standard items DNA is respectively GCK Rs2244164 wild types DNA (gene order is referring to NCBI dbSNP database NC_000007.14), HK2rs656489 are wild Type DNA (gene order is referring to NCBI dbSNP database NC_000002.12), HIF1- α rs10136168 wild type DNA (bases Because sequence is referring to NCBI dbSNP database NC_000014.9).
In a preferred embodiment of the kit that the present invention is provided, the ultra-pure water is the water of HPLC ranks.
The present invention also provides primer sets as described above and prepared for detecting GCK genes, HK2 genes and HIF1- α genes Application in the reagent of parting.
The primer sets and reagent of the detection nasopharyngeal carcinoma glycolysis related gene parting provided compared to prior art, the present invention Box, which has the advantages that, to be
First, compared with conventional nasopharyngeal carcinoma diagnosis method, primer sets and kit of the invention have quick, sensitive and spy The features such as opposite sex is good, can carry out risk assessment, to take necessary prevention, early diagnosis early to control etc. and to arrange to human nasopharyngeal carcinoma in time in early stage Apply；
2nd, by combining multiple PCR technique, Single base extension technology and matrix solid-dispersion flight time Analytical technique of mass spectrum carries out Genotyping detection, with testing result is accurate, specificity is high, detection cycle is short, detection method is clever Quick advantage；
3rd, by being provided with positive reference substance and negative controls in kit so that the kit is in detection sugar During glycolysis related gene GCK, HK2 and HIF1- α Genotypings, the accuracy of testing result can be preferably ensured.
Brief description of the drawings
Fig. 1 is the sequencer map of clinical sample GCK rs2244164 sites wild type；
Fig. 2 is the sequencer map of clinical sample GCK rs2244164 site mutation heterozygous；
Fig. 3 is the homozygous sequencer map of clinical sample GCK rs2244164 site mutations；
Fig. 4 is the sequencer map of clinical sample HK2rs656489 sites wild type；
Fig. 5 is the sequencer map of clinical sample HK2rs656489 site mutation heterozygous；
Fig. 6 is the homozygous sequencer map of clinical sample HK2rs656489 site mutations；
Fig. 7 is the sequencer map of clinical sample HIF1- α rs10136168 sites wild type；
Fig. 8 is the sequencer map of clinical sample HIF1- α rs10136168 site mutation heterozygous；
Fig. 9 is the homozygous sequencer map of clinical sample HIF1- α rs10136168 site mutations.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, below in conjunction with the accompanying drawings and embodiment, it is right The present invention is described in further detail.It should be appreciated that specific embodiment described herein is only to explain the present invention, and It is not used in the restriction present invention.
First, experimental subjects
This clinical research strictly observes conspicuous Bielski declaration, and has obtained the attached tumour hospital of Xiangya Medical College, Zhongnan Univ The approval of Ethics Committee.
The inclusive criteria of seminar's case：
1) choose in October, 2014 in July, 2016 the attached tumour hospital of Xiangya Medical College, Zhongnan Univ be in hospital and warp Histopathology is diagnosed as 723 patients of nasopharyngeal carcinoma.
2) not by other treatments such as radiation and chemotherapies before recruiting.
3) previously without nasopharyngeal carcinoma and other malignant neoplastic disease histories.
4) 6 months before blood specimen collection are without blood transfusion history.
5) the receiving all survey related to this research and contribution venous blood are agreed to.
6) agree to fill in《Informed consent form》.
7) all subjects consanguinity-less relation to each other.
The inclusive criteria of control group：
1) 857 the normal residents, epidemic disease that the selection same period checks UP in Changsha Grade A hospital hospital
Screening results are learned without any malignant tumour medical history.
2) Hunan Province's resident population.
3) the receiving all survey related to this research and contribution venous blood are agreed to.
4) all research objects, which are agreed, fills in《Informed consent form》.
2nd, experimental program
Case-control study is carried out, case group and healthy control group is devised.Using Mass ARRAY systems, pass through detection The approach of peripheral blood cells, is detected to the SNP of 3 key genes of glycolytic pathway：GCK rs2244164 sites (gene Sequence is referring to NCBI dbSNP database NC_000007.14), (gene order is referring to NCBI dbSNP in HK2rs656489 sites Database NC_000002.12), (gene order is referring to NCBI dbSNP databases NC_ in HIF1- α rs10136168 sites 000014.9).Utilize genotype point between the matching degree of Haploview, SPSS18.0 detection Hardy-Weinberg balances, group Cloth compares.
3rd, experimental method
3.1 clinical samples are collected：
According to entering to arrange standard collection Nasopharyngeal Carcinoma Patients and normal medical examiner's venous blood 2-5ml, EDTA anti-freezings are used, are used Omega kits extract DNA and are stored in -20 DEG C of refrigerators.
With MassARRAy Sequenom, (Bio Miao Biological Technology (Beijing, China) are public Department) time-of-flight mass spectrometry biochip system to it is all enter group objects carry out GCK rs2244164, HK2rs656489 and HIF1- The Genotyping in α rs10136168 sites, analyzes the correlation with nasopharyngeal carcinoma risk.
3.2DNA extract：
DNA extraction kit (Omega USA) extracts DNA from whole blood, illustrates to operate in strict accordance with kit.Specific step It is rapid as follows：
1) blood sample in EDTA anticoagulant tubes is overturned and be sufficiently mixed several times；
2) whole blood is added in 15ml centrifuge tubes, and adds the cell pyrolysis liquid Buffer NL of 2.5 times of volumes, up and down It is reverse 5 times, cell is fully cracked；The consumption of all cushioning liquid is added according to table 2.
3) 2000 × g is centrifuged 5 minutes.Slow supernatant discarding, centrifuge tube is upside down on clean blotting paper 2 minutes, is protected Card precipitation is always in centrifuge tube.
4) buffer solution XL and protease OB mixed liquor is configured according to table 1.The mixed liquor of 0.5 times of volume is added, immediately whirlpool Rotation 10 seconds is until precipitation distribution substantially uniformity.
5) 65 DEG C of water-baths 10-30 minutes, during which notice whether observation precipitation dissolves completely, and it is blackish green molten to become clarification Liquid.
6) isopropanol is added when clear ink green solution is down to room temperature, overturns and fully mix to the white thread or cluster of appearance The genomic DNA of shape.
7) 2000 × g is centrifuged 5 minutes.Slow supernatant discarding.Centrifuge tube is upside down on clean blotting paper until current To the greatest extent.
8) 70% ethanol solution is added, the 10s that turns upside down, which is mixed, cleans genomic DNA.
9) 2000 × g is centrifuged 3 minutes.Slow supernatant discarding.Centrifuge 1 minute, washed out by again.Centrifuge tube is inverted On clean blotting paper 10-15 minutes until the ethanol volatilization in pipe is complete.
10) 200-500 μ l DNA lysate EB Buffer are added into centrifuge tube, are beaten DNA with buffer solution with robbing.
11) centrifuge tube is placed in 65 DEG C of water-bath and be incubated -1 hour 10 minutes, it is ensured that DNA is completely molten in the process Solution.
12) application trace dna concentration analyzer determines the OD values of sample, records DNA concentration, is placed in -20 DEG C of environment guarantors Deposit.
Various buffering capacities needed for the different volumes blood of table 1
3.3 gene polymorphism sites are selected：
Using Biotechnology Information and the mononucleotide polymorphic database of world HapMap plan database national centers, I Obtained the Hans GCK, HK2, IDH1 and HIF-1 α genes and upstream and downstream 500kb information.Screening criteria is r2＜ 0.8, Minimum gene frequency is more than 5% (in Chinese Han Population), is Tag SNP.
3.4 genetic polymorphism detections and analysis：
3.4.1 design of primers and synthesis
According to SNP site sequence information, designed using the primer-design software Assay design3.1 of sequenom companies PCR reacts and single base extends primer, referring to table 2.
The primer sets of the cancer glycolysis related gene parting of table 2
3.4.2 DNA is extracted
Using finished product kit, the DNA in blood sample, tissue, cell, saliva is extracted.Use NanoDrop2000 instruments (Beijing Cole's moral scientific ＆ trading Co., Ltd.) carries out the detection of OD values, and 1.25% agarose gel electrophoresis detection, DNA quality inspections are qualified, turn Tong is to 96 orifice plates, and -20 DEG C store for future use.
3.4.3 Sequenom MassArray system genes typing step
A) pcr amplification reaction
1) principle
PCR (Polymerase chain reaction, PCR), which is used to expand, is located at two ends known array Between region of DNA domain.Each PCR cycle, reaction mixture is all different with three kinds of primer extend in template denaturation, primer annealing At a temperature of sequentially incubate.The process can be used a kind of programmable temperature thermal cycler and reach automation.
2. step
1) take and PCR premixed liquids (PCR master mix) are configured in 1.5mlEP pipes, and vibrate low-speed centrifugal.Reactive component, Referring to table 3：
The PCR master mix of table 3 react related reagent formulation components
Remarks：MgCl2Ultimate density is 3.5mM, 1.875mM in wherein PCR Buffer, and MgCl2Itself with the addition of 1.625mM。
2) the road Tong liquid devices of 8 Dao Huo 12 are selected, 4 μ l PCR master mix are added in each well of 384 orifice plates, The mixing of 1 μ l template DNAs (20ng/ul) is eventually adding, 384 hole shrouding films are carefully covered, and fastens each hole, PCR programs are prevented When the phenomenon such as occur evaporating.1000rpm centrifuges 1minute.
3) following pcr amplification reaction system is set, referring to table 4.PCR reaction plates are positioned in PCR instrument, startup program.
The pcr amplification reaction system of table 4
B) product alkaline phosphatase treatment
1) after PCR reactions terminate, by PCR primer SAP (Shrimp alkaline phosphatase, shrimp alkalescence phosphorus Sour enzyme) processing, with remove system middle reaches from dNTPs.
2) alkaline phosphatase treatment reaction solution, SAP Mix reactive components, referring to table 5 are prepared in new 1.5mlEP pipes：
The SAP Mix reactive components of table 5
SAP mix reagents Concentration Volume (1rxn)
Water, HPLC ranks NA 1.53μl
SAP Buffer 10X 0.17μl
SAP enzymes 1U/ul 0.30μl
Amount to - 2.00μl
3) SAP mix are added into 384 hole PCR reaction plates, for each alkaline phosphatase treatment reacting hole, reacts cumulative volume For 7ul, wherein PCR primer 5ul, SAP mix 2ul.
4) after the completion of Tong liquid, 384 hole shrouding films are carefully covered, and fasten each hole, prevent occurring evaporating during PCR programs Following response procedures are carried out after phenomenon, centrifugation.
5) SAP response procedures are set：37℃20min；85℃5min；4℃∞.And 384 hole reaction plates are positioned over PCR instrument On, startup program.
C) single base extension
1) after alkaline phosphatase treatment terminates, single base extension, the μ l of reaction system cumulative volume 9 are carried out.
2) single base extension liquid, EXTEND Mix reactive components, referring to table 6 are prepared in new 1.5mlEP pipes：
The EXTEND Mix reactive components of table 6
EXTEND Mix reagents Concentration (μ l of in 9) Volume (1rxn)
Water, HPLC ranks NA 0.619μl
iPLEX Buffer Plus 0.222X 0.200μl
IPLEX termination mixs 1X 0.200μl
(7 μM of primer sets：14μM) 0.625uM：1.25uM 0.940μl
IPLEX enzymes 1X 0.041μl
Amount to - 2.0μl
Remarks：(7μM：14 μM) it is expressed as the double concentration of high-quality primer sets.It is i.e. low in final 9 μ l concentration-response liquid Quality primer sets concentration is 0.625uM and high-quality primer sets concentration is 1.25uM.
3) EXTEND Mix correspondences are added into 384 hole reaction plates.For each reacting hole, single base extension system, Referring to table 7：
The single base extension system of table 7
Reagent Volume (ul)
EXTEND Mix 2
SAP+PCR reaction solutions 7
Amount to 9
4) after the completion of Tong liquid, 384 hole shrouding films are carefully covered, and fasten each hole, prevent from occurring during PCR journeys evaporating etc. existing As carrying out following response procedures after centrifugation.
5) extension program is set, referring to table 8：
The extension program of table 8
D) purifying resin
1) in 384/6MG Dimple plates uniform potting resin and place dry it within 10 minutes.
2) in the μ L water of each Kong Zhongjia 16 of 384 sample planes.
3) 384 sample planes are gently turned and be buckled on Dimple plates, then rapping makes resin fall into the every of sample plane In individual hole.
4) 384 sample planes are placed into room temperature rotation in upset centrifuge to mix 30 minutes.
E) chip point sample
Start MassARRAY Nanodispenser RS1000 point sample instruments, by the extension products Tong after purifying resin extremely On 384-well SpectroCHIP bioarray.
F) Mass Spectrometer Method and data output
SpectroCHIP chips after point sample are analyzed using MALDI-TOF mass spectrographs (German Bruker companies), detection As a result initial data and Genotyping figure are obtained using TYPER4.0 softwares, checks the integrality and correctness of data file, will As a result respective stored medium is saved into, bioinformatic analysis is then carried out.
4th, interpretation of result
UsingThe softwares of SPSS Statistics 18, HAPLOVIEW4.2 softwares.Hardy-Weinberg is balanced Model is used to detection sample genotype and gene frequency.Pearson ' s Chi-square Tests or Fisher ' s accurately examine (when When there is any expecterd frequency less than 5) it is used to compare genotype and the genotypes distribution and allele frequencies between case group and control group.Meter Hazard ratio (Odds ratios, OR) and 95% credibility interval (Confidence intervals, CI) is calculated to assess each genotype With the relative risk of allele.
Contrasted for each genotype with reference gene type, and classify by sex and carried out subgroup analysis.Group Between Clinical symptoms data detected using Chi-square Test.It is considered as statistically significant when p value is less than 0.05.
As a result it is as follows：
1st, distribution of the significant SNP site in case group and control group, referring to table 9.
Distribution of the SNP site of table 9 in case group and control group
HIF-1a rs10136168 are the mononucleotide polymorphics positioned at upstream region of gene, it is a discovery of the invention that in the site, phase For wild type GG and mutant homozygous type GA crowd, the crowd for carrying mutation heterozygous GA is the group of people at high risk for suffering from nasopharyngeal carcinoma, 95% credibility interval 1.042-1.618.
2nd, distribution of the SNP site in male's subgroup, referring to table 10.
Distribution of the SNP site of table 10 in male's subgroup
In male's subgroup, between each genotype distribution in HIF1- α rs17113978 sites in each crowd, system is found no Meter learns the research conclusion of meaning.
3rd, distribution of the SNP site in women subgroup, referring to table 11.
Distribution of the SNP site of table 11 in women subgroup
Found in women subgroup, GCK rs2244164 saltant type homozygotes CC, which has, significantly suffers from nasopharyngeal carcinoma excessive risk, Its risk is 1.97 times of unmutated crowd, 95% credibility interval 1.067-3.653.This mononucleotide polymorphic is located at Portugal The introne of glucokinase encoding gene, with enhancing sub-feature, may accelerate glycolysis path.Dashed forward in HK 2rs656489 Modification allele A is ill protection sexual factor, and risk is 0.655 times of unmutated crowd, 95% credibility interval 0.449-0.985.It is located at the noncoding region that gene 5 ' is held, may and transfer stable to regulation transcription, mRNA it is related by This, above mononucleotide polymorphic may make a significant impact on Warburg effects.
In addition, sequencing result figure is referring to Fig. 1-Fig. 9.Wherein Fig. 1-3 is respectively that clinical sample GCK rs2244164 sites are wild The sequencer map of raw type, mutation heterozygous and mutant homozygous type；Fig. 4-6 is respectively that clinical sample HK2 rs656489 sites are wild The sequencer map of type, mutation heterozygous and mutant homozygous type；Fig. 7-9 is respectively that clinical sample HIF1- α rs10136168 sites are wild The sequencer map of type, mutation heterozygous and mutant homozygous type.Above-mentioned sequencing result is true and reliable, meets clinical detection to Genotyping Standard requirement.
The primer sets and kit for the detection nasopharyngeal carcinoma glycolysis related gene parting that the present invention is provided have following beneficial Effect is
First, compared with conventional nasopharyngeal carcinoma diagnosis method, primer sets and kit of the invention have quick, sensitive and spy The features such as opposite sex is good, can carry out risk assessment, to take necessary prevention, early diagnosis early to control etc. and to arrange to human nasopharyngeal carcinoma in time in early stage Apply；
2nd, by combining multiple PCR technique, Single base extension technology and matrix solid-dispersion flight time Analytical technique of mass spectrum carries out Genotyping detection, with testing result is accurate, specificity is high, detection cycle is short, detection method is clever Quick advantage；
3rd, by being provided with positive reference substance and negative controls in kit so that the kit is in detection sugar During glycolysis related gene GCK, HK2 and HIF1- α Genotypings, the accuracy of testing result can be preferably ensured.
Embodiments of the invention are the foregoing is only, are not intended to limit the scope of the invention, it is every to utilize this hair The equivalent flow conversion that bright description is made, or other related technical fields are directly or indirectly used in, similarly wrap Include in the scope of patent protection of the present invention.
SEQUENCE LISTING
<110>Wang Hui
<120>Detect the primer sets and kit of nasopharyngeal carcinoma glycolysis related gene parting
<130> 2016
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 30
<212> DNA
<213>Artificial sequence
<400> 1
acgttggatg cataatgtgt tttctccgcc 30
<210> 2
<211> 30
<212> DNA
<213>Artificial sequence
<400> 2
acgttggatg gggacttact cctgatctac 30
<210> 3
<211> 30
<212> DNA
<213>Artificial sequence
<400> 3
acgttggatg ctggtgtcta gcattagctg 30
<210> 4
<211> 30
<212> DNA
<213>Artificial sequence
<400> 4
acgttggatg tgatcttgga catgggatgg 30
<210> 5
<211> 30
<212> DNA
<213>Artificial sequence
<400> 5
acgttggatg tcaccctctg atgcagttac 30
<210> 6
<211> 30
<212> DNA
<213>Artificial sequence
<400> 6
acgttggatg cagctctgtt ctttactgcc 30

Claims (6)

1. a kind of primer sets for detecting nasopharyngeal carcinoma glycolysis related gene parting, it is characterised in that the glycolysis related gene Respectively GCK genes, HK2 genes and HIF1- α genes, the GCK genes, HK2 genes and HIF1- α gene polynorphisms site Respectively GCK rs2244164, HK2 rs656489 and HIF1- α rs10136168, the primer sets include：
(1) for GCK rs2244164 sites,
Amplimer is：
GCK rs2244164 forward direction amplimers：
5’-ACGTTGGATGCATAATGTGTTTTCTCCGCC-3’；
The reverse amplimers of GCK rs2244164：
5’-ACGTTGGATGGGGACTTACTCCTGATCTAC-3’；
(2) for HK2rs656489 sites,
Amplimer is：
HK2 rs656489 forward direction amplimers：
5’-ACGTTGGATGCTGGTGTCTAGCATTAGCTG-3’；
The reverse amplimers of HK2 rs656489：
5’-ACGTTGGATGTGATCTTGGACATGGGATGG-3’；
(3) for HIF1- α rs10136168 sites,
Amplimer is：
HIF1- α rs10136168 forward direction amplimers：
5’-ACGTTGGATGTCACCCTCTGATGCAGTTAC-3’；
The reverse amplimers of HIF1- α rs10136168：
5’-ACGTTGGATGCAGCTCTGTTCTTTACTGCC-3’。
2. a kind of kit for detecting nasopharyngeal carcinoma glycolysis related gene parting, it is characterised in that the kit includes containing The positive amplimers of GCK rs2244164 as claimed in claim 1 are expanded with reverse amplimer, HK2 rs656489 forward directions Primer and reverse amplimer, the positive amplimers of HIF1- α rs10136168 and reverse amplimer, PCR Buffer, MgCl2, dNTP and Taq enzyme PCR premixed liquids.
3. kit according to claim 2, it is characterised in that each reagent component proportioning is in the PCR premixed liquids：
4. kit according to claim 3, it is characterised in that the kit also includes：Positive reference substance and feminine gender Reference substance, the positive reference substance is saltant type standard items DNA, and the negative controls are wild type standard items DNA.
5. kit according to claim 3, it is characterised in that the ultra-pure water is the water of HPLC ranks.
6. the primer sets described in claim 1 are preparing the examination for detecting GCK genes, HK2 genes and HIF1- α Genotypings Application in agent.
CN201611240834.5A 2016-12-29 2016-12-29 Primer group and kit for detecting glycolytic related genotyping of nasopharyngeal carcinoma Active CN107058486B (en)

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CN108998527A (en) * 2018-08-25 2018-12-14 右江民族医学院附属医院 A kind of relevant osteopontin functionality SNP of nasopharynx cancer onset risk and application
CN110331198A (en) * 2019-03-27 2019-10-15 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） It is a kind of for the SNP marker of tumor prognosis and its application
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