CN104178555A - A kit for detecting esophagus cancer related risky genes - Google Patents
A kit for detecting esophagus cancer related risky genes Download PDFInfo
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- CN104178555A CN104178555A CN201310194887.8A CN201310194887A CN104178555A CN 104178555 A CN104178555 A CN 104178555A CN 201310194887 A CN201310194887 A CN 201310194887A CN 104178555 A CN104178555 A CN 104178555A
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Abstract
A kit for detecting esophagus cancer related risky genes is disclosed. The kit includes: specific primer pairs, specific fluorescence probe pairs, common components for fluorogenic quantitative PCR detection; and the like, wherein the specific primer pairs and the specific fluorescence probe pairs are used for simultaneously detecting a number rs2274223 SNP site on a PLCE1 gene, a number rs13042395 SNP site on a C20orf54 gene, a number rs2074356 SNP site on a C12orf51 gene, a number rs2014300 SNP site on an RUNX1 gene, a number rs11066015 SNP site on an ACAD10 gene and a number rs10052657 SNP site on a PDE4D gene. The kit evaluates the risk of esophagus cancer for a person through simultaneously detecting single nucleotide polymorphic site genotypes of the PLCE1, the C20orf54, the C12orf51, the RUNX1, the ACAD10 and the PDE4D which are closely related with the esophagus cancer.
Description
Technical field
The present invention relates to molecular biology and medical field, more specifically, the present invention relates to a kind of test kit that detects esophagus cancer relevant risk gene, assess individuality and suffer from the risk of esophagus cancer by detect the mononucleotide polymorphism site genotype of gene PLCE1, C20orf54, C12orf51, RUNX1, ACAD10 and the PDE4D associated with esophagus cancer simultaneously.
Background technology
The esophageal carcinoma is one of modal six large malignant tumours in the world, in Cancer in China associated death reason, ranked fourth position.Esophageal canceration is considered to a multistage, progressive evolution, and in this process, environmental factors and inherited genetic factors all play a part very important.The effect of various carcinogenic factors in environment, can make the structure and function of esophageal epithelial cell DNA change, in the different steps of this change procedure, may there is several genes to change, and act on different steps, different biological modifications may occur, the two more or less exists different impacts each other, and inflammation is considered to swollen neoplastic promotor, chronic inflammatory diseases has increased the risk of cell carcinogenesis, and provides a kind of microenvironment for neoplastic process.
At present, the full gene association analysis method of scientists utilization (GWAS), has found out six esophageal carcinoma tumor susceptibility gene: PLCE1, C20orf54, C12orf51, RUNX1, ACAD10 and PDE4D.
PLCE1 gene is positioned on No. ten karyomit(e), it belongs to Phospholipid hydrolase family a member, coding phosphor lipase albumen, and Phospholipid hydrolase albumen has been played the part of a pivotal player in Cellular Signaling Transduction Mediated, by catalysis phosphatidylinositols 4,5-bisphosphate produces second messenger's InsP3 (inositol-1,4,5-triphosphate, and diacylglycerol (diacylglycerol IP3), DAG), the latter can cause the overexpression of protein kinase C (PKC), and further combined with the target protein of the isozyme of the PKC much being represented by PKC.These products can cause that the thin intramicellar reaction of some row participates in growth, differentiation, apoptosis and the vasculogenesis of cell, and genetic expression.PLCE1 gene is also in the news and promotes the development of skin and intestinal canal tumour to form by inflammation signal path.
C20orf54 is riboflavin translocator encoding gene, and the major function of its protein product is that riboflavin is proceeded in cell by extracellular.C20orf54 genovariation may be to cause riboflavin transhipment abnormal, affects the major reason that riboflavin utilizes.Riboflavin deficiency can increase the risk that esophageal squamous cell carcinoma occurs.C20orf54, as mankind's riboflavin transport vehicle, regulate the absorption of riboflavin at enteron aisle, and riboflavin plays an important role to the stable of intracellular environment.C12orf51 is No. 12 open reading frames of karyomit(e), relevant to acetaldehyde and carcinogens metabolism, Cell apoptosis and proliferation etc.
RUNX1 is one of member in RUNX transcription factor protein family, and RUNX1 is very important transcription factor, expression that can two-ways regulation hematopoiesis genes involved.The adjustable expression that comprises the hematopoiesis genes involveds such as φt cell receptor β chain, medullary system peroxidase B, interleukin-3, granulocyte-macrophage colony stimutaing factor of RUNX1.In the promotor of these genes, all contain PEBP2 sequence, RUNX1 is with it in conjunction with rear transcriptional activation.RUNX1 albumen can receive multiple posttranslational modification, comprises phosphorylation, acetylize, methylates, glycosylation and ubiquitination, and its activity can be subject to the impact of these posttranslational modifications, thereby regulates differentiation, apoptosis and the self of hematopoietic cell.Research shows, the low expression of RUNX1 may be that the important biomolecule that development occurs malignant tumour is learned mark.
ACAD10 is positioned karyomit(e) 12q24.1 section No. 12, has 21 exon compositions, exon and the nearly 71kbp of intron sequences span on karyomit(e).ACAD10 has a typical fatty acyl-CoA dehydrogenase conserved domain, and this is a functional domain at fatty acyl-CoA dehydrogenase family (ACADs) high conservative.By carrying out structural domain sequence alignment with other ACADs family members, we find that ACAD10 comprises nearly all completely conservative amino acid sites that is considered in ACADs family member completely or is close to.
PDE4D, signal factor in the cell that cAMP is various kinds of cell, the albumen mass-energy of this genes encoding reduces the level of cAMP.
Research represents, in said gene, specific gene locus has accumulation correlation with esophageal carcinoma risk, carries dangerous variation more, and the risk of the generation esophageal carcinoma is larger.The risk of carrying the raw esophageal carcinoma of human hair of 2 to 6 risk genes types is increased to 4.60 by 1.27 gradually.
Summary of the invention
6 SNPs loci polymorphisms based on PLCE1, C20orf54, C12orf51, RUNX1, ACAD10 and PDE4D gene can be used to assessment individuality and suffer from the basis of esophagus cancer risk, the invention provides a kind of test kit that detects esophagus cancer relevant risk gene.
Test kit comprises:
The Auele Specific Primer that detects rs2274223 SNP Genetic polymorphism type on PLCE1 gene to and specificity fluorescent probe pair;
The Auele Specific Primer that detects rs13042395 SNP Genetic polymorphism type on C20orf54 gene to and specificity fluorescent probe pair;
The Auele Specific Primer that detects rs2074356 SNP Genetic polymorphism type on C12orf51 gene to and specificity fluorescent probe pair;
The Auele Specific Primer that detects rs2014300 SNP Genetic polymorphism type on RUNX1 gene to and specificity fluorescent probe pair;
The Auele Specific Primer that detects rs11066015 SNP Genetic polymorphism type on ACAD10 gene to and specificity fluorescent probe pair;
The Auele Specific Primer that detects rs10052657 SNP Genetic polymorphism type on PDE4D gene to and specificity fluorescent probe pair;
Quantitative fluorescent PCR reaction component (comprises Taq enzyme, dNTP mixed solution, MgCl
2solution, reaction buffer, deionized water etc.).
Auele Specific Primer described in this test kit designs referring to for rs10052657 SNP site on rs11066015 SNP site and PDE4D gene on rs2014300 SNP site, ACAD10 gene on rs2074356 SNP site, RUNX1 gene on rs13042395 SNP site, C12orf51 gene on rs2274223 SNP site, C20orf54 gene on PLCE1 gene, and energy specific amplification goes out to comprise the primer pair of the DNA fragmentation in these 6 SNPs sites.Designing this class primer pair is that those skilled in the art can be unlabored.Preferably, in test kit, comprise there is SEQ ID NO:1 and 2, the primer pair of sequence shown in 3 and 4,5 and 6,7 and 8,9 and 10,11 and 12.Auele Specific Primer synthesizes the synthetic technology of available routine.Those skilled in the art will appreciate that primer of the present invention is not limited to this six pairs of primers.
Specificity fluorescent probe described in this test kit designs referring to for rs10052657 SNP site on rs11066015 SNP site and PDE4D gene on rs2014300 SNP site, ACAD10 gene on rs2074356 SNP site, RUNX1 gene on rs13042395 SNP site, C12orf51 gene on rs2274223 SNP site, C20orf54 gene on PLCE1 gene, can go out by fluorescent quantitative PCR technique specific detection the Taqman probe pair of these six SNPs loci gene types.Designing this class probe is that those skilled in the art can be unlabored.Preferably, in test kit, comprise there is SEQ ID NO:13 and 14, the Taqman probe pair of sequence shown in 15 and 16,17 and 18,19 and 20,21 and 22,23 and 24.Specificity fluorescent probe synthesizes the probe synthetic technology of available routine.Those skilled in the art can understand, specificity fluorescent Taqman probe of the present invention is to being not limited to this six pairs of probes, all can be used for fluorescence quantitative PCR method detect described in the present invention, be used for individual six the SNPs sites of suffering from esophagus cancer risk of assessment probe all within the scope of the present invention.
Component and the content of test kit of the present invention comprise:
5 μ l 10 X-fluorescence quantitative PCR reaction buffers,
0.5 μ l 25mM dNTP mixed solution,
3 μ l 25mM MgCl
2solution,
0.125 μ l (5units/ μ is Taq archaeal dna polymerase l),
20 μ M Auele Specific Primers are to every each 0.225 μ l of primer,
10 μ M specificity fluorescent probes are to every each 0.25 μ l of probe,
Deionized water 26.625 μ l.
This test kit detects application for a person-portion, and the storage temperature of test kit is-20 DEG C.
Embodiment:
Below in conjunction with specific embodiment, further set forth the present invention.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, or the condition of advising according to manufacturer.
The use of embodiment 1. detection kit
The extraction of step 1:DNA template
Gather human peripheral blood with vacuum test tube, extract the genomic dna in blood.
Step 2: quantitative fluorescent PCR reaction
Use can detect the fluorescent quantificationally PCR detecting kit of esophagus cancer relevant risk gene, wherein, comprises following primer pair and fluorescent probe pair:
Sense primer 1:5 '-ATGTGGAACGAGC – 3 ' (SEQ ID NO:1)
Antisense primer 1:5 '-AAATACAAGATCA – 3 ' (SEQ ID NO:2)
Sense primer 2:5 '-GTTCTGACCAGGG – 3 ' (SEQ ID NO:3)
Antisense primer 2:5 '-GAGGAGATGGCCC – 3 ' (SEQ ID NO:4)
Sense primer 3:5 '-GCTGGTAAACAGT – 3 ' (SEQ ID NO:5)
Antisense primer 3:5 '-CTGTGGTCTGGTG – 3 ' (SEQ ID NO:6)
Sense primer 4:5 '-GTGAGCATTAAAA – 3 ' (SEQ ID NO:7)
Antisense primer 4:5 '-GTCAGTATAGGGA – 3 ' (SEQ ID NO:8)
Sense primer 5:5 '-GCATCATCCCTGG – 3 ' (SEQ ID NO:9)
Antisense primer 5:5 '-CTGTGGGGAGGGC – 3 ' (SEQ ID NO:10)
Sense primer 6:5 '-CGAAGATTGTTCC – 3 ' (SEQ ID NO:11)
Antisense primer 6:5 '-ATTATCTCTCTCC – 3 ' (SEQ ID NO:12)
Band VIC fluorophor probe 1:5 '-TGTTCCaCGTTC-3 ' (SEQ ID NO:13)
Band FAM fluorophor probe 1:5 '-TGTTCCgCGTTC-3 ' (SEQ ID NO:14)
Band VIC fluorophor probe 2:5 '-CACCGTcATTGT-3 ' (SEQ ID NO:15)
Band FAM fluorophor probe 2:5 '-CACCGTtATTGT-3 ' (SEQ ID NO:16)
Band VIC fluorophor probe 3:5 '-TCAGATaTGAAC-3 ' (SEQ ID NO:17)
Band FAM fluorophor probe 3:5 '-TCAGATgTGAAC-3 ' (SEQ ID NO:18)
Band VIC fluorophor probe 4:5 '-CAGGGCaACCAC-3 ' (SEQ ID NO:19)
Band FAM fluorophor probe 4:5 '-CAGGGCgACCAC-3 ' (SEQ ID NO:20)
Band VIC fluorophor probe 5:5 '-CCACTCaATGAC-3 ' (SEQ ID NO:21)
Band FAM fluorophor probe 5:5 '-CCACTCgATGAC-3 ' (SEQ ID NO:22)
Band VIC fluorophor probe 6:5 '-TAGTTTaCAGTC-3 ' (SEQ ID NO:23)
Band FAM fluorophor probe 6:5 '-TAGTTTcCAGTC-3 ' (SEQ ID NO:24)
Sense primer 1, antisense primer 1, with VIC fluorophor probe 1, with FAM fluorophor probe 1 specifically for the rs2274223 SNP loci polymorphism detecting on PLCE1 gene;
Sense primer 2, antisense primer 2, with VIC fluorophor probe 2, with FAM fluorophor probe 2 specifically for the rs13042395 SNP loci polymorphism detecting on C20orf54 gene;
Sense primer 3, antisense primer 3, with VIC fluorophor probe 3, with FAM fluorophor probe 3 specifically for the rs2074356 SNP loci polymorphism detecting on C12orf51 gene;
Sense primer 4, antisense primer 4, with VIC fluorophor probe 4, with FAM fluorophor probe 4 specifically for the rs2014300 SNP loci polymorphism detecting on RUNX1 gene;
Sense primer 5, antisense primer 5, with VIC fluorophor probe 5, with FAM fluorophor probe 5 specifically for the rs11066015 SNP loci polymorphism detecting on ACAD10 gene;
Sense primer 6, antisense primer 6, with VIC fluorophor probe 6, with FAM fluorophor probe 6 specifically for the rs10052657 SNP loci polymorphism detecting on PDE4D gene;
2 independently quantitative fluorescent PCR reactions are carried out respectively in 6 SNPs sites, the system of each reaction is cumulative volume 10 μ l, and comprising concentration is DNA profiling 2 μ l, 1 μ l 10 X-fluorescence quantitative PCR reaction buffers, 0.1 μ l 25mM dNTP mixed solution, the 0.6 μ l 25mM MgCl of 20ng/ μ l
2(5units/ μ is the sense primer of Taq archaeal dna polymerase, 20 μ M and the band VIC fluorescent probe of the each 0.225 μ l of antisense primer, 10 μ M and the each 0.25 μ l of band FAM fluorescent probe l), deionized water 5.325 μ l for solution, 0.025 μ l.
On ABI9700 type pcr amplification instrument, react, reaction conditions is 50 DEG C, 2 minutes, 95 DEG C, 10 minutes, and 95 DEG C, 30 seconds of carrying out 60 circulations, 60 DEG C, 1 minute.After finishing, reaction reads fluorescent amount on ABI7900 type quantitative real time PCR Instrument.
Step 3:SNP gene type assay
The those skilled in the art that are familiar with fluorescent quantitative PCR technique can, by the final sample fluorescence volume showing on identification quantitative real time PCR Instrument, can determine the genotype in detected SNP site according to the power of different sequence probe VIC and FAM fluorescent signal.
Embodiment 2. carries out the service of esophagus cancer relevant risk gene test to patient
Step 1:DNA extracts
By hospital laboratory doctor, examinee is carried out the collection of peripheral blood, adopt vacuum test tube to gather peripheral blood, and extract genomic dna wherein.
Step 2: genotype tests
Use test kit provided by the invention, fluorescence quantitative PCR detection is carried out respectively in rs2014300 SNP site on rs13042395 SNP site on rs2274223 SNP site, C20orf54 gene on the PLCE1 gene of examinee's genomic dna, rs2074356 SNP site, RUNX1 gene on C12orf51 gene, rs11066015 SNP site on ACAD10 gene and the rs10052657 SNP site on PDE4D gene, determine the genotype in these six SNPs sites.
Step 3: individuality is suffered from the analysis of esophagus cancer risk
By to the genotypic analysis of detected person SNPs, provide the analysis report list of suffering from esophagus cancer risk.In report, describe the height that detected person suffers from esophagus cancer risk in detail, and describe and understand to detected person the analysis report list of suffering from esophagus cancer risk by doctor in detail.
Claims (6)
1. a test kit that detects esophagus cancer relevant risk gene, is characterized in that: comprise detect on PLCE1 gene on rs2274223 SNP site, C20orf54 gene on rs13042395 SNP site, C12orf51 gene on rs2074356 SNP site, RUNX1 gene on rs2014300 SNP site, ACAD10 gene on rs11066015 SNP site and PDE4D gene simultaneously the Auele Specific Primer in rs10052657 SNP site to and specificity fluorescent probe to, Taq enzyme, dNTP mixed solution, MgCl
2solution, quantitative fluorescent PCR reaction buffer, deionized water.
2. test kit according to claim 1, it is characterized in that: described Auele Specific Primer designs referring to for rs10052657 SNP site on rs11066015 SNP site and PDE4D gene on rs2014300 SNP site, ACAD10 gene on rs2074356 SNP site, RUNX1 gene on rs13042395 SNP site, C12orf51 gene on rs2274223 SNP site, C20orf54 gene on PLCE1 gene, energy specific amplification goes out to comprise the primer pair of the DNA fragmentation in these 6 SNPs sites.
3. test kit according to claim 1, it is characterized in that: described specificity fluorescent probe designs referring to for rs10052657 SNP site on rs11066015 SNP site and PDE4D gene on rs2014300 SNP site, ACAD10 gene on rs2074356 SNP site, RUNX1 gene on rs13042395 SNP site, C12orf51 gene on rs2274223 SNP site, C20orf54 gene on PLCE1 gene, can go out by fluorescent quantitative PCR technique specific detection the Taqman probe pair of these 6 SNPs loci gene types.
4. test kit according to claim 1, is characterized in that: contained Auele Specific Primer has SEQ ID NO:1 and 2 to being selected from, the primer pair of sequence shown in 3 and 4,5 and 6,7 and 8,9 and 10,11 and 12.
5. test kit according to claim 1, is characterized in that: contained specificity fluorescent probe be selected from there is SEQ ID NO:13 and 14, the Taqman probe pair of sequence shown in 15 and 16,17 and 18,19 and 20,21 and 22,23 and 24.
6. test kit according to claim 1, is characterized in that: the component of test kit and content comprise 10 X-fluorescence quantitative PCR reaction buffers, 0.5 μ l 25mM dNTP mixed solution, 3 μ l 25mM MgCl
2solution, 0.125 μ l (5units/ μ l) Taq archaeal dna polymerase, 20 μ M Auele Specific Primers to every each 0.225 μ l of primer, 10 μ M specificity fluorescent probes to every the each 0.25 μ l of probe, deionized water 26.625 μ l, this test kit detects application for a person-portion, and the storage temperature of test kit is-20 DEG C.
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| CN201310194887.8A CN104178555A (en) | 2013-05-23 | 2013-05-23 | A kit for detecting esophagus cancer related risky genes |
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| CN201310194887.8A CN104178555A (en) | 2013-05-23 | 2013-05-23 | A kit for detecting esophagus cancer related risky genes |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104593499A (en) * | 2015-01-20 | 2015-05-06 | 汕头大学医学院 | Detection kit for carrying out efficient molecular subtyping on esophageal cancer susceptible gene loci |
| CN106755468A (en) * | 2017-01-13 | 2017-05-31 | 中山大学 | A kind of primer and its PCR method of detection RUNX1rs2268277 gene pleiomorphisms |
| CN106811519A (en) * | 2016-08-17 | 2017-06-09 | 上海易瑞生物科技有限公司 | A kind of oesophagus cancer susceptibility gene detection and genotyping kit and its application |
| CN110218793A (en) * | 2019-06-03 | 2019-09-10 | 郑州大学第一附属医院 | A kind of SNP marker relevant to cancer of the esophagus auxiliary diagnosis |
-
2013
- 2013-05-23 CN CN201310194887.8A patent/CN104178555A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104593499A (en) * | 2015-01-20 | 2015-05-06 | 汕头大学医学院 | Detection kit for carrying out efficient molecular subtyping on esophageal cancer susceptible gene loci |
| CN104593499B (en) * | 2015-01-20 | 2017-12-05 | 汕头大学医学院 | A kind of detection kit of the efficient molecule parting of cancer of the esophagus susceptibility loci |
| CN106811519A (en) * | 2016-08-17 | 2017-06-09 | 上海易瑞生物科技有限公司 | A kind of oesophagus cancer susceptibility gene detection and genotyping kit and its application |
| CN106755468A (en) * | 2017-01-13 | 2017-05-31 | 中山大学 | A kind of primer and its PCR method of detection RUNX1rs2268277 gene pleiomorphisms |
| CN110218793A (en) * | 2019-06-03 | 2019-09-10 | 郑州大学第一附属医院 | A kind of SNP marker relevant to cancer of the esophagus auxiliary diagnosis |
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