CN107043821A - Primer and detection method for detecting the related SNP site of cutaneum carcinoma neurological susceptibility - Google Patents
Primer and detection method for detecting the related SNP site of cutaneum carcinoma neurological susceptibility Download PDFInfo
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Abstract
The invention discloses the primer for detecting the related SNP site of cutaneum carcinoma neurological susceptibility, include primer, the primer in rs801114 sites, the primer in rs11170164 sites and the primer in rs2151280 sites in rs7538876 sites.Using the method for the primer detection cutaneum carcinoma neurological susceptibility associated SNP positions, comprise the following steps：(1) gather sample and extract DNA；(2) PCR amplifications, the glue reclaim of target gene are carried out respectively using four kinds of primers；(3) concentration of glue reclaim product is determined, fluorescence labeling PCR is carried out and purifies；(4) machine in purified product is sequenced, and SNP testing results is analyzed.The primer specificity is good, sensitivity is high, accuracy is good, and detection method is simple, and predictable person under inspection suffers from the risk of cutaneum carcinoma.
Description
Technical field
The invention belongs to biological technical field, and in particular to for detecting drawing for the related SNP site of cutaneum carcinoma neurological susceptibility
Thing and detection method.
Background technology
The result of cutaneum carcinoma system epidermal keratinocytes neoplasm, is one of common malignant tumour of the mankind.White
The incidence of disease is high in kind people, and its incidence of disease highest is basal-cell carcinoma, next to that the squamous cell carcinoma easily shifted, pernicious
Melanoma occupies the 3rd, and the most common cutaneum carcinoma of Chinese is basal-cell carcinoma, and southern area is higher than northern area, daylight
Ultraviolet in irradiation is the most common risk factor of basal-cell carcinoma, wherein 85% basal-cell carcinoma betides face and neck
Portion.With our people's growth in the living standard, the requirement of increasing people, especially Ms to autologous skin is also increasingly
It is high.Because cutaneum carcinoma easily betides face and neck, disfeatured or other influences if may be brought by operative treatment.Pass through inspection
Survey the SNP related to cutaneum carcinoma neurological susceptibility, it becomes possible to help cutaneum carcinoma Susceptible population to understand its risk for suffering from cutaneum carcinoma, do in advance
Good prevention work, it is to avoid the generation of cutaneum carcinoma.SNP (SNP) detects, because its polymorphism information amount is big, have
Distribution is wide, quantity is more, hereditary capacity is stable, it is easy to the features such as batch-automated detection, at present as long after restriction fragment
Degree polymorphism (RFLP) mark and microsatellite are the third generation genetic marker after simple tandem sequence repeats (STR) mark.For reality
The early of existing cutaneum carcinoma is found, early prevented and early control, and cutaneum carcinoma disease susceptibility has been detected by SNP (SNP)
As study hotspot both domestic and external, there is genome-wide association study to show, rs7538876, rs801114, rs11170164,
Four SNP sites such as rs2151280 and cutaneum carcinoma disease susceptibility are closely related, and it is located at PADI6 on human chromosomal 1p36
The rs7538876 neurological susceptibilities equipotential of gene is A；Rs801114 neurological susceptibilities equipotential on human chromosomal 1q42 is G；It is located at
The rs11170164 neurological susceptibilities equipotential of KRT5 genes is A；The rs2151280 of CDKN2A/B genes on human chromosomal 9p21
Neurological susceptibility equipotential is C.Therefore, the polymorphism of the detection SNP site related to cutaneum carcinoma neurological susceptibility is passed through, it can be estimated that suffer from skin
The susceptible risk of cancer, guidance is provided for the prevention from suffering from the diseases of cutaneum carcinoma.But detection and cutaneum carcinoma disease susceptibility were not reported at present
The primer of closely related aforementioned four SNP site.
The content of the invention
For above-mentioned deficiency of the prior art, the invention provides the SNP position related for detecting cutaneum carcinoma neurological susceptibility
The primer and detection method of point, its primer specificity are good, and detection method accuracy is high, available for the risk assessment of cutaneum carcinoma, is
The prevention from suffering from the diseases of cutaneum carcinoma provides guidance.
To achieve the above object, the technical solution adopted for the present invention to solve the technical problems is：
Primer for detecting the related SNP site of cutaneum carcinoma neurological susceptibility, these primers include drawing for rs7538876 sites
Thing, the primer in rs801114 sites, the primer in rs11170164 sites and the primer in rs2151280 sites；
Wherein, the primer in rs7538876 sites is F：5′-CCTTTCTACATAGCCATTC-3′(SEQ ID No：1) and
R：5′-CTTAATGTAACCAGGAGACC-3′(SEQ ID No：2)；
The primer in rs801114 sites is F：5′-GATTACGAAAGGGAAGAG-3′(SEQ ID No：And R 3)：5′-
GCTATCCAGTATGAAAACAC-3′(SEQ ID No：4)；
The primer in rs11170164 sites is F：5′-GAGGATATCCATCAGCAC-3′(SEQ ID No：And R 5)：5′-
GAGGCAAACTTATTGTTGAG-3′(SEQ ID No：6)；
The primer in rs2151280 sites is F：5′-TGAACTCCTTTCACTATACC-3′(SEQ ID No：And R 7)：5′-
GGACAGAATGTCTTAGTGTAG-3′(SEQ ID No：8).
Using the method for the related SNP site polymorphism of above-mentioned primer detection cutaneum carcinoma neurological susceptibility, comprise the following steps：
(1) gather sample and extract its genomic DNA；
(2) PCR for being carried out target gene to genomic DNA respectively using above-mentioned four kinds of primers is expanded, and to amplified production
Carry out glue reclaim；
(3) concentration mensuration is carried out to glue reclaim product, then calculation template amount enters performing PCR amplification (fluorescence labeling reaction)
And purify；
(4) it is sequenced on GeXP automatic sequencers (Beckman companies of the U.S.), with software (GenomeLabTM GeXP
(Genetic Analysis System)) SNP testing results are analyzed.
Further, PCR amplification system is in step (2)：DNA profiling 100-150ng, concentration are 5pmol/ μ L forward direction
The μ L of primer 3.0, concentration is the 5pmol/ μ L μ L of 3.0 μ L, Prime STAR MAX of reverse primer 25.0, uses ddH2O supplies volume
To 50 μ L.
Further, pcr amplification reaction condition is in step (2)：98 DEG C of pre-degenerations 2min, 98 DEG C of denaturation 30s, 58 DEG C are moved back
Fiery 15s, 72 DEG C of extension 15s, carry out 30 circulations, last 72 DEG C of extension 5min are after 4 DEG C of preservations.
Further, PCR amplification system is in step (3)：The μ L of DTCS Master Mix 2.5, concentration is 5pmol/ μ L
Reverse primer 1.0 μ L, DNA profiling 20ng, use ddH2O supplies volume to 10 μ L.
Further, pcr amplification reaction condition is in step (3)：94 DEG C of pre-degenerations 30s, 94 DEG C of denaturation 25s, 55 DEG C are moved back
Fiery 25s, 60 DEG C of extension 3min, 30 circulations, last 60 DEG C of extensions 20min.
Provided by the present invention for the primer and detection method of the related SNP site of detection cutaneum carcinoma neurological susceptibility, with
Lower beneficial effect：
(1) the invention provides the primer for detecting skin cancer susceptibility gene related locus, the primer has specificity
The good advantage of high, accuracy, enters performing PCR amplification according to the primer pair target gene, people can be accurately determined by direct Sequencing
Pleomorphism site genotype in skin cancer susceptibility gene, can predict that the risk that person under inspection suffers from cutaneum carcinoma is several according to genotype results
Rate, and targetedly take effective precautionary measures to be prevented, evade as far as possible or postpone the sick generation, this is to cutaneum carcinoma
Prediction and prevention be significant.
(2) present invention also offers a kind of side using specific primer detection cutaneum carcinoma neurological susceptibility associated SNP positions
Method, the detection method is simple, and its testing result also has specificity good, and sensitivity is high, the advantages of accuracy is good.
Brief description of the drawings
Fig. 1 is to enter the agarose gel electrophoresis figure that performing PCR expands products therefrom to primer；
Fig. 2 is the sequencing result figure that rs7538876 loci polymorphisms are detected；
Fig. 3 is the sequencing result figure that rs801114 loci polymorphisms are detected；
Fig. 4 is the sequencing result figure that rs11170164 loci polymorphisms are detected；
Fig. 5 is the sequencing result figure that rs2151280 loci polymorphisms are detected；
Fig. 6 is the fluorescence proof diagram of rs7538876 loci polymorphism testing results；
Fig. 7 is the fluorescence proof diagram of rs801114 loci polymorphism testing results；
Fig. 8 is the fluorescence proof diagram of rs11170164 loci polymorphism testing results；
Fig. 9 is the fluorescence proof diagram of rs2151280 loci polymorphism testing results.
Embodiment
Embodiment 1 determines the SNP related to cutaneum carcinoma neurological susceptibility
The document of the genome-wide association study related to cutaneum carcinoma neurological susceptibility, final screening are searched in ncbi database
Go out 4 good SNP of correlation, including：Rs7538876, rs801114, rs11170164, rs2151280.Pass through NCBI data
The sequence of this 4 SNP sites of library lookup, and drawn with reference to the result of study of document：The PADI6 bases on human chromosomal 1p36
The rs7538876 neurological susceptibilities equipotential of cause is A；Rs801114 neurological susceptibilities equipotential on human chromosomal 1q42 is G；It is located at
The rs11170164 neurological susceptibilities equipotential of KRT5 genes is A；The rs2151280 of CDKN2A/B genes on human chromosomal 9p21
Neurological susceptibility equipotential is C.
The primer of the design amplification SNP site of embodiment 2
Using the sequence of above-mentioned SNP site as template, primer is designed with Primer-BLAST, by comparing the several of influence primer
Individual key factor, filters out relatively good primer sequence, including：
The primer in rs7538876 sites is F：5′-CCTTTCTACATAGCCATTC-3′(SEQ ID No：And R 1)：5′-
CTTAATGTAACCAGGAGACC-3′(SEQ ID No：2)；
The primer in rs801114 sites is F：5′-GATTACGAAAGGGAAGAG-3′(SEQ ID No：And R 3)：5′-
GCTATCCAGTATGAAAACAC-3′(SEQ ID No：4)；
The primer in rs11170164 sites is F：5′-GAGGATATCCATCAGCAC-3′(SEQ ID No：And R 5)：5′-
GAGGCAAACTTATTGTTGAG-3′(SEQ ID No：6)；
The primer in rs2151280 sites is F：5′-TGAACTCCTTTCACTATACC-3′(SEQ ID No：And R 7)：5′-
GGACAGAATGTCTTAGTGTAG-3′(SEQ ID No：8).
These primer sequences are synthesized, it is standby that the primer dry powder of synthesis is diluted to 5pmol/ μ l.PCR is passed through to primer
Amplification, is verified, electrophoretogram is shown in Fig. 1 through agarose gel electrophoresis, wherein, 1,2,3 holes expand bar for the primer in rs7538876 sites
Band；4th, 5,6 holes are the primer amplified band in rs801114 sites；7th, 8,9 holes are the primer amplified band in rs11170164 sites；
10th, 11,12 holes are the primer amplified band in rs2151280 sites.
As shown in Figure 1, purpose band is clear, without miscellaneous band, and clip size and design is in the same size, illustrates that designs draws
Thing specificity is good.
The detection of the SNP site polymorphism related to cutaneum carcinoma neurological susceptibility of embodiment 3
(1) extraction of genomic DNA
Person under inspection peripheral blood 5mL is gathered with EDTA anticoagulant tubes, the extracting method of its genomic DNA is with reference to poba gene group
Operating procedure in the specification of DNA extraction kit (being purchased from Beijing Tiangeng biochemical technology Co., Ltd), by specification is carried out
Extract.The complete genome DNA of acquisition using ultramicron nucleic acid-protein analyzer (cypress essence BioDrop μ Lite) detect its concentration with
And purity, record initial data.Concentration and purity are all reached that the DNA of requirement is placed in -20 DEG C of refrigerators and saved backup.
(2) PCR amplifications, electrophoresis and the glue reclaim of target gene
PCR is expanded：Use above-mentioned rs7538876, rs801114, the primer difference in rs11170164 and rs2151280 sites
Performing PCR amplification is entered to the DNA of extraction.Reaction system is 50 μ L, and specific as shown in table 1, amplification program is as shown in table 2.PCR reacts
After the completion of, take out 4 DEG C of PCR primer and preserve and (need -20 DEG C of placement to freeze overnight).
The PCR reaction systems of table 1
| Reagent | Volume (μ L) |
| DNA | a(100-150ng) |
| ddH2O | 19-a |
| Primer F | 3.0 |
| Primer R | 3.0 |
| Primer STAR MAX | 25.0 |
| total | 50.0 |
The PCR amplification programs of table 2
After PCR amplifications terminate, 50 μ L amplified productions are taken, using 1% agarose gel electrophoresis, electrophoretic parameters are voltage
Specific band nothing but in 120V, time 30min, electrophoresis result；Cut after the completion of electrophoresis under gel electrophoresis images analyzer
Gel containing purpose fragment, gel piece is fitted into EP pipes, and the method for glue reclaim is with reference to TaKaRa MiniBEST Agarose
The specification of Gel DNA Extraction Kit Ver.4.0 kits, finally carries out concentration mensuration, 4 to the product of glue reclaim
DEG C save backup.
(3) mark, purify and be sequenced
Mark：A clean 0.2mL PCR pipe is taken, is sequentially added：DTCS Master Mix, sequencing primer, sterilizing are super
The DNA concentration that the consumption of pure water, template DNA, wherein template DNA and sterilizing ultra-pure water is determined by previous step is determined, is added
Template DNA amount be 20ng, reaction system is 10 μ L, and specific then to carry out fluorescence labeling PCR as shown in table 3, amplification program is shown in
Table 4；
The PCR reaction systems of table 3
| Reagent | Volume (μ L) |
| DTCS Master Mix | 2.5 |
| Primer R | 1.0 |
| ddH2O | 6.5-b |
| Template DNA | b(20ng) |
| Amount to | 10.0 |
The PCR amplification programs of table 4
Purifying：It is 3mol/L, sodium-acetate buffer, 0.1mol/L of the pH value for 5.2 by concentration, pH value is 8.0
Na2Edta buffer liquid and Glycogen are using volume ratio as 2:2:1 prepares terminate liquid, then takes 5 μ L terminate liquids to add above-mentioned fluorescence
Fully mixed in mark reaction PCR primer, centrifuge, liquid is transferred in a new 1.5mL EP pipe, 50 μ L are then added
Precooling absolute ethyl alcohol, is fully mixed, and is put into -20 DEG C of refrigerator freezing 10min and is centrifuged 5min after 12000r/min, supernatant discarding,
The ethanol of 150 μ L precoolings 70% is added into EP pipes again, supercentrifuge centrifugation is put into, 12000r/min centrifugation 2min are discarded
Clearly, slightly centrifuge, remaining liquid in EP pipes is blotted with pipettor, EP lids are opened, room temperature is dried to white precipitate bleach, then
25 μ L SLS (Sample Loading Solution) dissolving DNA is added into EP pipes, 8min, brief centrifugation is stood；
Sequencing：The DNA of dissolving is added in 96 hole sample plate holes, bubble can not be produced during addition, is then added again
Enter appropriate mineral oil；One piece of 96 new orifice plate is taken, 10 are added in corresponding aperture and drips dissociating buffer, ultra-pure water is added into glue groove
To liquid close to instruction line, finally 96 hole sample panels, 96 hole buffer solutions and glue groove loading GeXP sequenators are sequenced.
Pass through sequence analysis software (GenomeLabTMGeXP (Genetic Analysis System)), by sequencing result
It is compared with standard sequence, SNP site is found, by the type for analyzing base at SNP site, it is possible to obtain SNP site
Genotype.
2-1,2-2 and 2-3 are the sequencing result figure of rs7538876 loci polymorphisms detection in Fig. 2, and genotype is followed successively by
GG, AG and AA；Wherein GG is the non-susceptible genotype in rs7538876 sites, and AG and AA is the easy sensillary base in rs7538876 sites
Because of type.
3-1,3-2 and 3-3 are the sequencing result figure of rs801114 loci polymorphisms detection in Fig. 3, and genotype is followed successively by
TT, TG and GG；Wherein TT is the non-susceptible genotype in rs801114 sites, and TG and GG is the tumor susceptibility gene in rs801114 sites
Type.
4-1,4-2 and 4-3 are the sequencing result figure that rs11170164 loci polymorphisms are detected in Fig. 4, and genotype is followed successively by
GG, AG and AA；Wherein GG is the non-susceptible genotype in rs11170164 sites, and AG and AA is the susceptible of rs11170164 sites
Genotype.
5-1,5-2 and 5-3 are the sequencing result figure that rs2151280 loci polymorphisms are detected in Fig. 5, and genotype is followed successively by
TT, TC and CC；Wherein TT is the non-susceptible genotype in rs2151280 sites, and TC and CC is the easy sensillary base in rs2151280 sites
Because of type.
Above-mentioned sequencing result figure illustrates to detect SNP using PCR sequencing PCR without phenomenons such as set peak, background peaks, miscellaneous peak, the peaks that floats
Point polymorphism, its specificity is good, accuracy is high.Therefore, testing agency can according to the polymorphism of above-mentioned SNP site, assess by
Inspection person suffers from the risk of cutaneum carcinoma, and guidance is provided for the prevention from suffering from the diseases of cutaneum carcinoma.
The checking of the SNP site polymorphic detection result related to cutaneum carcinoma neurological susceptibility of embodiment 4
6-1,6-2 and 6-3, which are, in Fig. 6 is verified to above-mentioned testing result using quantitative fluorescent PCR sonde method
The fluorescence proof diagram of rs7538876 loci polymorphism testing results, genotype is followed successively by GG, AG and AA；In Fig. 7 7-1,7-2 and
7-3 is the fluorescence proof diagram of rs801114 loci polymorphism testing results, and genotype is followed successively by TT, TG and GG；8-1,8- in Fig. 8
2 and 8-3 is the fluorescence proof diagram of rs11170164 loci polymorphism testing results, and genotype is followed successively by GG, AG and AA；In Fig. 9
9-1,9-2,9-3 are the fluorescence proof diagram of rs2151280 loci polymorphism testing results, and genotype is followed successively by TT, TC and CC, on
State fluorescence probe method testing result consistent with PCR sequencing PCR testing result 100%, further demonstrate and detect SNP using PCR sequencing PCR
The specificity and accuracy of point polymorphism.
SEQUENCE LISTING
<110>Co., Ltd of medical test institute of Chengdu Zhong Chuanqing sections
<120>Primer and detection method for detecting the related SNP site of cutaneum carcinoma neurological susceptibility
<130> 2016
<160> 8
<170> PatentIn version 3.3
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Claims (6)
1. the primer for detecting the related SNP site of cutaneum carcinoma neurological susceptibility, it is characterised in that the primer includes
The primer in rs7538876 sites, the primer in rs801114 sites, the primer in rs11170164 sites and rs2151280 sites
Primer；
Wherein, the primer in rs7538876 sites is F：5 '-CCTTTCTACATAGCCATTC-3 ' and R：5′-
CTTAATGTAACCAGGAGACC-3′；
The primer in rs801114 sites is F：5 '-GATTACGAAAGGGAAGAG-3 ' and R：5′-
GCTATCCAGTATGAAAACAC-3′；
The primer in rs11170164 sites is F：5 '-GAGGATATCCATCAGCAC-3 ' and R：5′-
GAGGCAAACTTATTGTTGAG-3′；
The primer in rs2151280 sites is F：5 '-TGAACTCCTTTCACTATACC-3 ' and R：5′-
GGACAGAATGTCTTAGTGTAG-3′。
2. using the method for the related SNP site polymorphism of the primer detection cutaneum carcinoma neurological susceptibility described in claim 1, its feature
It is, comprises the following steps：
(1) gather sample and extract its genomic DNA；
(2) PCR for being carried out target gene to genomic DNA respectively using above-mentioned four kinds of primers is expanded, and amplified production is carried out
Glue reclaim；
(3) concentration mensuration is carried out to glue reclaim product, then calculation template amount carries out fluorescence labeling PCR and purify；
(4) machine in the purified product in step (3) is sequenced, and SNP testing results is analyzed.
3. the method for the related SNP site polymorphism of detection cutaneum carcinoma neurological susceptibility according to claim 2, its feature exists
In PCR amplification system is in step (2)：DNA profiling 100-150ng, concentration are the 5pmol/ μ L μ L of forward primer 3.0, concentration
For the 5pmol/ μ L μ L of 3.0 μ L, Prime STAR MAX of reverse primer 25.0, ddH is used2O supplies volume to 50 μ L.
4. the method for the related SNP site polymorphism of detection cutaneum carcinoma neurological susceptibility according to claim 2, its feature exists
In pcr amplification reaction condition is in step (2)：98 DEG C of pre-degenerations 2min, 98 DEG C of denaturation 30s, 58 DEG C of annealing 15s, 72 DEG C of extensions
15s, carries out 30 circulations, last 72 DEG C of extensions 5min.
5. the method for the related SNP site polymorphism of detection cutaneum carcinoma neurological susceptibility according to claim 2, its feature exists
In PCR amplification system is in step (3)：The μ L of DTCS Master Mix 2.5, concentration is the 5pmol/ μ L μ of reverse primer 1.0
L, DNA profiling 20ng, uses ddH2O supplies volume to 10 μ L.
6. the method for the related SNP site polymorphism of detection cutaneum carcinoma neurological susceptibility according to claim 2, its feature exists
In pcr amplification reaction condition is in step (3)：94 DEG C of pre-degenerations 30s, 94 DEG C of denaturation 25s, 55 DEG C of annealing 25s, 60 DEG C of extensions
3min, 30 circulations, last 60 DEG C of extensions 20min.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108531602A (en) * | 2018-05-29 | 2018-09-14 | 成都中创清科医学检验所有限公司 | A kind of primer and detection method for detecting the relevant SNP site of lymph cancer neurological susceptibility |
| CN108570503A (en) * | 2018-05-29 | 2018-09-25 | 成都中创清科医学检验所有限公司 | A kind of primer and its detection method for detecting chronic lymphocytic leukemia gene polymorphism sites |
| CN108588215A (en) * | 2018-05-03 | 2018-09-28 | 成都中创清科医学检验所有限公司 | A kind of primer and its detection method for detecting the relevant SNP site of familial hypercholesterolemia neurological susceptibility |
| CN109837349A (en) * | 2019-01-22 | 2019-06-04 | 橙狐(上海)信息科技有限责任公司 | A kind of Primer composition, kit and detection method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120122698A1 (en) * | 2008-07-07 | 2012-05-17 | Decode Genetics Ehf. | Genetic Variants Predictive of Cancer Risk in Humans |
| CN105821143A (en) * | 2016-05-26 | 2016-08-03 | 成都中创清科医学检验所有限公司 | Primer and method for detecting hypertension-susceptibility-related SNP site |
| CN105821147A (en) * | 2016-05-26 | 2016-08-03 | 成都中创清科医学检验所有限公司 | Primer and method for detecting rectal-cancer-susceptibility-related SNP site |
-
2017
- 2017-04-26 CN CN201710283252.3A patent/CN107043821A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120122698A1 (en) * | 2008-07-07 | 2012-05-17 | Decode Genetics Ehf. | Genetic Variants Predictive of Cancer Risk in Humans |
| CN105821143A (en) * | 2016-05-26 | 2016-08-03 | 成都中创清科医学检验所有限公司 | Primer and method for detecting hypertension-susceptibility-related SNP site |
| CN105821147A (en) * | 2016-05-26 | 2016-08-03 | 成都中创清科医学检验所有限公司 | Primer and method for detecting rectal-cancer-susceptibility-related SNP site |
Non-Patent Citations (2)
| Title |
|---|
| SIMON N STACEY等人: ""Common variants on 1p36 and 1q42 are associated with cutaneous basal cell carcinoma but not with melanoma or pigmentation traits"", 《NATURE GENETICS》 * |
| WILLIAM CS CHO、南娟等人: ""癌症相关遗传变异的新进展及其在诊断研发中的意义"", 《中国肺癌杂志》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108588215A (en) * | 2018-05-03 | 2018-09-28 | 成都中创清科医学检验所有限公司 | A kind of primer and its detection method for detecting the relevant SNP site of familial hypercholesterolemia neurological susceptibility |
| CN108531602A (en) * | 2018-05-29 | 2018-09-14 | 成都中创清科医学检验所有限公司 | A kind of primer and detection method for detecting the relevant SNP site of lymph cancer neurological susceptibility |
| CN108570503A (en) * | 2018-05-29 | 2018-09-25 | 成都中创清科医学检验所有限公司 | A kind of primer and its detection method for detecting chronic lymphocytic leukemia gene polymorphism sites |
| CN109837349A (en) * | 2019-01-22 | 2019-06-04 | 橙狐(上海)信息科技有限责任公司 | A kind of Primer composition, kit and detection method |
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Application publication date: 20170815 |