CN107937525A - NRAS mutation detection kits and extension primer based on Luminex - Google Patents
NRAS mutation detection kits and extension primer based on Luminex Download PDFInfo
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Abstract
The invention discloses a kind of NRAS genetic tests specific primer and liquid-phase chip, which mainly includes：For No. 12 codons on 2 exons of NRAS genes, No. 13 codons, 18 mutator types in No. 146 codons on the 61st bit codon, 4 exons and wild-type genotype on 3 exons separately design ASPE extension primers, there is that anti tag sequences are coated, have the magnetic bead of different colours coding；The primer of target sequence detect, with corresponding mutational site is needed for amplifying.NRAS gene mutation detection liquid-phase chips of the present invention can be used for 18 kinds of mutation types of detection NRAS genes, and detection site is more comprehensive, there is provided result it is relatively reliable, provide more accurate diagnosis basis for doctor.
Description
Technical field
The invention belongs to biology field, is related to medicine and biotechnology, relates particularly to a kind of NRAS genes
Mutation detection kit and extension primer, using liquid-phase chip technology detection and the relevant NRAS gene mutations of cancer.
Background technology
Ras genes are a kind of proto-oncogenes, and on the message bang path of cell, predominantly activation controls genetic transcription
Kinases, adjusts propagation and the differentiation of cell, is an important Downstream regulatory gene in EGFR signal transduction pathways.Normal thin
In born of the same parents, RAS exists with its non-activated state (GDP mating types), and when being subject to exogenous signals to stimulate, phosphorylation, conformational change occur for RAS
And as the GTP mating types of activation, the GTP-RAS activation downstream RAF signal transducers of activation, until being degraded.RAS genes
Oncogenicity point mutation can reduce the ability that RAS proteolysis GTP is GDP, cause RAS albumen to be combined with GTP continuation, under causing
The activation of path continuation is swum, cell is produced the malignant proliferation independent of growth factor, causes the generation of cancer.
Nerve-cell tumor rat meat knurl oncogene (Neuroblastoma ras viral oncogene homolog, NRAS)
It is one of Ras gene family members.The mutation of NRAS genes is predominantly located at the 2nd, 3, on 4 extrons, cause Several Kinds of Malignancy,
Mutation rate in colorectal cancer (colorectal cancer, CRC) patient is 1-6%, in chromoma (malignant
Melanoma the mutation probability in) is 20%, is oncogene common in human tumor.Clinical research finds NRAS mutation
Colorectal cancer patients are significantly reduced to the EGFR monoclonal antibody medicine sensitiveness based on Cetuximab, therefore NRAS gene mutations are
Anti-epidermal growth factor receptor monoclonal antibody treats the independent predictor of curative effect.NRAS genetic tests are classified as large intestine by European and American areas
The routine inspection that must be done before cancer patient's medical treatment, american cancer integrated network (NCCN)《The practice of NCCN colorectal cancer clinicals refers to
South》With《NCCN carcinoma of the rectum clinical practice guidelines》It is classified as clinical application essential items for inspection.In addition, also there is relevant research to show NRAS
Mutation is related with the TKI drug resistances in lung cancer therapy.Also clinical data shows that the inhibitor using Cdk4 as drug targets presses down with MEK
Agents use can treat the melanoma patients of NRAS mutation.To sum up, more and more evidences are shown, NRAS mutation exist
It is of great significance in the occurrence and development of many tumours such as human melanoma, colorectal cancer, lung cancer.Therefore, correlation is being carried out
In treatment of cancer, detection patient with the presence or absence of relevant NRAS gene functions mutation, can Accurate Prediction molecular targeted agents control
The validity for the treatment of, easy to clinical accurate medication, significantly improves curative effect of medication, patient is at utmost benefited, and can also avoid not
The waste of Medical Expense for Patients burden and social medical resource caused by the rational use of medicines.
Currently, the kit for being directed to people's NRAS detection in Gene Mutation is based substantially on Fluorescence PCR assay platform development, most
More mutation type detections also only have 9 kinds.It is special that although fluorescent PCR with liquid-phase chip technology is respectively provided with high throughput, high sensitivity etc.
Point, the advantages that detection cycle can be shortened, cost is reduced and improve efficiency.Before this, the present inventor researched and developed NRAS bases to PCR
Because of mutation detection specific primer and liquid-phase chip (ZL2011102697755), its site detected includes 2 extra of NRAS genes
13 kinds of gene mutation types above aobvious son, 3 exons, 4 exons.And in practical clinical, find above-mentioned detection
Site is not met by the needs of single tube complete detection analysis.
The content of the invention
An object of the present invention is to provide NRAS gene mutation detection liquid-phase chips, which can be used for detecting
2 exons of NRAS genes, 3 exons, on 4 exons thus with colorectal cancer, non-small cell lung cancer, maligna
The relevant 18 kinds of gene mutation type, that is, G34A, G34T of plain knurl, G34C, G35A, G35C, G35T, G37C, G37T, G38A,
G38T, G38C, C181A, A182C, A182T, A182G, A183T, G436A, G437T and normal three kinds of genotype, there is provided more
Relatively comprehensively, accurate, easily detection mode, overcomes current single tube detection single reaction, detection site is single, and sensitivity is not
The shortcomings that height, testing cost is high, strong evidence is provided for clinical treatment.
Realize that above-mentioned purpose technical solution is as follows.
A kind of NRAS gene mutation detection liquid-phase chips kit, includes：
(A) is for No. 12 codons on 2 exons of NRAS genes, No. 13 codons, and the on 3 exons
18 mutator types in No. 146 codons and wild-type genotype on 61 bit codons, 4 exons separately design
ASPE extension primers, every kind of ASPE extension primers are directed to the specific primer sequence in mutational site by the tag sequences and 3 ' ends at 5 ' ends
Row composition, the specific primer sequence of mutator type are selected from following at least one：For the SEQ ID in G34A sites
NO.22, for the SEQ ID NO.23 in G34T sites, for the SEQ ID NO.24 in G34C sites, for the SEQ in G35A sites
ID NO.25, for the SEQ ID NO.26 in G35C sites, for the SEQ ID NO.27 in G35T sites, for G37C sites
SEQ ID NO.28, for the SEQ ID NO.29 in G37T sites, for the SEQ ID NO.30 in G38A sites, for G38T
The SEQ ID NO.31 of point, for the SEQ ID NO.32 in G38C sites, for the SEQ ID NO.33 in C181A sites, for
The SEQ ID NO.34 in A182C sites, for the SEQ ID NO.35 in A182T sites, for the SEQ ID in A182G sites
NO.36, for the SEQ ID NO.37 in A183T sites, for the SEQ ID NO.38 in G436A sites, for G437T sites
SEQ ID NO.39；
The ASPE extension primers of wild-type genotype are selected from corresponding following at least one：For the SEQ ID of 2 exons
NO.40, for 3 exon SEQ ID NO.41, for 4 exon SEQ ID NO.73；
(B) has the magnetic bead that anti-tag sequences are coated, there are different colours to encode, the anti-tag sequences and magnetic
Spacer sequence is additionally provided among pearl connection；The anti-tag sequences are selected from SEQ ID NO.42~SEQ ID NO.62, and
The anti-tag sequences can be matched correspondingly with tag sequences complete complementary selected in (A)；
(C) is used to amplify the primer for needing target sequence detect, with corresponding mutational site.
In one of the embodiments, the amplimer is selected from following at least a pair of：For the SEQ of 2 exons
ID NO.63 and SEQ ID NO.64, for 3 exon SEQ ID NO.65 and SEQ ID NO.66, for 4 exons
SEQ ID NO.67 and SEQ ID NO.68.
In one of the embodiments, the corresponding ASPE for including 18 described in claim 1 mutational sites prolongs
Extend thing, the magnetic bead and the amplimer.
In one of the embodiments, the spacerarm is 5-10 T.
It is a further object to provide a kind of ASPE extension primers for NRAS detection in Gene Mutation.
Realize that the technical solution of above-mentioned purpose is as follows.
For the ASPE extension primers of NRAS detection in Gene Mutation liquid, for No. 12 on 2 exons of NRAS genes
Codon, No. 13 codons, the 61st bit codon on 3 exons, 18 in No. 146 codons on 4 exons
Mutator type and wild-type genotype separately design ASPE extension primers, and every kind of ASPE extension primers are by 5 ' the tag sequences held
Formed with specific primer sequence of the 3 ' ends for mutational site, the specific primer sequence of mutator type be selected from
Lower at least one：For the SEQ ID NO.22 in G34A sites, for the SEQ ID NO.23 in G34T sites, for G34C sites
SEQ ID NO.24, for the SEQ ID NO.25 in G35A sites, for the SEQ ID NO.26 in G35C sites, for G35T
The SEQ ID NO.27 in site, for the SEQ ID NO.28 in G37C sites, for the SEQ ID NO.29 in G37T sites, for
The SEQ ID NO.30 in G38A sites, for the SEQ ID NO.31 in G38T sites, for the SEQ ID NO.32 in G38C sites,
For the SEQ ID NO.33 in C181A sites, for the SEQ ID NO.34 in A182C sites, for the SEQ ID in A182T sites
NO.35, for the SEQ ID NO.36 in A182G sites, for the SEQ ID NO.37 in A183T sites, for G436A sites
SEQ ID NO.38, for the SEQ ID NO.39 in G437T sites；
The ASPE extension primers of wild-type genotype are selected from corresponding following at least one：For the SEQ ID of 2 exons
NO.40, for 3 exon SEQ ID NO.41, for 4 exon SEQ ID NO.73；
The tag sequences are selected from：SEQ ID NO.1—SEQ ID NO.21.
In one of the embodiments, the ASPE extension primers of mutator type in following at least one
Kind：For the sequence being made of SEQ ID NO.1 and SEQ ID NO.22 in G34A sites, for G34T sites by SEQ ID
The sequence of NO.2 and SEQ ID NO.23 compositions, for being made of SEQ ID NO.3 and SEQ ID NO.24 for G34C sites
Sequence, for the sequence being made of SEQ ID NO.4 and SEQ ID NO.25 in G35A sites, for G35C sites by SEQ
The sequence of ID NO.5 and SEQ ID NO.26 compositions, is made of for G35T sites SEQ ID NO.6 and SEQ ID NO.27
Sequence, for the sequence being made of SEQ ID NO.7 and SEQ ID NO.28 in G37C sites, for G37T sites by
The sequence of SEQ ID NO.8 and SEQ ID NO.29 compositions, for G38A sites by SEQ ID NO.9 and SEQ ID NO.30
The sequence of composition, for the sequence being made of SEQ ID NO.10 and SEQ ID NO.31 in G38T sites, for G38C sites
The sequence being made of SEQ ID NO.11 and SEQ ID NO.32, for C181A sites by SEQ ID NO.12 and SEQ
The sequence of ID NO.33 compositions, for the sequence being made of SEQ ID NO.13 and SEQ ID NO.34 in A182C sites, for
The sequence being made of SEQ ID NO.14 and SEQ ID NO.35 in A182T sites, for A182G sites by SEQ ID
The sequence of NO.15 and SEQ ID NO.36 compositions, is made of for A183T sites SEQ ID NO.16 and SEQ ID NO.37
Sequence, for the sequence being made of SEQ ID NO.17 and SEQ ID NO.38 in G436A sites, for G437T sites
The sequence being made of SEQ ID NO.18 and SEQ ID NO.39；
Corresponding at least one of the ASPE extension primers described in wild-type genotype in following：For 2 exons
The sequence being made of SEQ ID NO.19 and SEQ ID NO.40, for 3 exons, by SEQ ID NO.20 and SEQ ID
The sequence of NO.41 compositions, for 4 exons, the sequence being made of SEQ ID NO.21 and SEQ ID NO.73.
NRAS gene mutation detection liquid-phase chips of the present invention mainly include the advantages of the following aspects：
1, in order to adapt to the needs of clinical detection, develops the detection in new site.Inventor is according to the warp accumulated before
Test, design be suitably used for 18 sites can abrupt climatic change parallel ASPE extension primers, make the specificity of primer extend and prolong
Efficiency raising is stretched, reduces non-specific extension.The suitable tag sequences of simultaneous selection, reduce and as far as possible avoid probe primer with
Cross reaction between anti-tag sequences；And the rational PCR amplification primer of design, can efficient amplification inspection piece,
Avoid non-specific amplification, and the stability of detecting system.
2. in liquid-phase chip technology of the present invention, use is selected to be applicable in the magnetic bead of MAGPIX, luminex instrument, generation
For being only capable of the microballoon that is detected in luminex instruments before, and corresponding technology has been carried out when designing primer and probe for this
Consider.
3rd, NRAS gene mutation detection liquid-phase chips of the present invention can be used for 18 kinds of mutation types of detection NRAS genes,
Detection site is more comprehensive, there is provided result it is relatively reliable, provide more accurate diagnosis basis for doctor.
Embodiment
Embodiment 1
One NRAS liquid-phase chips mainly include：
1ASPE primers
For No. 12 codons on 2 exon of NRAS genes, No. 13 codons, the 61st on 3 exons is close
Mutator type in No. 146 codons and wild-type genotype on numeral, 4 exons separately design ASPE and specifically extend
Primer.ASPE primer sequences are made of including sequence label TAG sequences and the primer specifically extended two parts.Primer sequence table is such as
Under：
Table one：NRAS gene mutation ASPE primer sequences
The tag sequences of two ASPE primer marks of table and the coated anti-tag sequence informations of magnetic bead
2：The coated magnetic beads of anti-tag
According to the selected tag sequences of the ASPE special primers of design, anti-tag sequence bags complementary therewith are selected
2 are shown in Table by the corresponding anti-tag sequences above magnetic bead surfaces, the magnetic bead label and magnetic bead of selection and its numbering.
The 21 kinds of magnetic beads selected in upper table, surface is coated in by anti-tag sequences, is connected between 5-10 T between them
Every arm sequence.The effect of spacerarm is that anti-tag sequences are spaced apart to and also ensured anti-tag sequences are put with magnetic bead surfaces
In hydrophilic environments.In addition, the spacer sequence can reduce steric hindrance, efficiency and the hybridization for improving hybridization reaction are anti-
Should specificity.3：Expand the primer in site to be measured
No. 12 codons, No. 13 codons of NRAS genes are respectively positioned on above 2 exons of NRAS genes, so being directed to
No. 12 codons, No. 13 codons and wild-type genotype can design primer, three kinds of genotype of 2 exons at both ends
Expanded, primer mark is EXON2 (NRAS-P1-F, NRAS-P1-R).No. 61 codons of NRAS genes are located at 3 extras and show
On son, the pcr amplification primer substance markers of wild type for 3 exons and saltant type design for EXON3 (NRAS-P2-F,
NRAS-P3-R).On 146 exon position, 4 exon of NRAS genes, designed for its wild type and saltant type
Pcr amplification primer substance markers be EXON4 (NRAS-P3-F, NRAS-P4-R).The sequence information of primer is as follows：
Three NARS gene magnification primer sequence tables of table
All primers are synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Every primer after synthesis is used respectively
10mmol/L Tris Buffer are configured to the storage liquid of 100pmol/mL.
Embodiment 2
Sample is detected using above-mentioned NRAS liquid-phase chips
First, the DNA extractions of sample：
Reference《Molecular cloning》On the correlation technique of DNA extractions, DNA to be detected is obtained.
2nd, the PCR amplification of sample to be tested
Amplification for NRAS gene types devises three pairs of primer sequences, respectively 2 exons for NRAS genes,
3 exons, 4 exons, amplified production size are respectively 198bp, 169bp, 161bp, and specific primer sequence information is shown in Table
Three.NRAS amplimer mixed liquors are configured first：Take 3 couples of primer storing liquid 100ul to add in 1.5ml centrifuge tubes respectively, mix
It is uniformly NRAS amplimer mixed liquors.Multi-PRC reaction system is as follows：
| PCR reaction systems | Often react (μ l) |
| NF water | 20.6 |
| 5×Colorless GoTaq Flexi Buffer | 10 |
| 25mM MgCl2 | 7 |
| NRAS amplimer mixed liquors | 6 |
| 2.5mM dNTP | 4 |
| GoTaq Hot Start polymerase(5U/μl) | 0.4 |
| Sample | 2 |
| Cumulative volume | 50 |
PCR reaction conditions are as follows：95℃3min；94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 40s, 30 circulations；72℃10min；4
DEG C save backup.
3rd, the digestion processing of PCR product
1. taking the product after 7.5ul PCR reactions, 10 × SAP of 1ul buffer solutions, 1ul SAP enzymes and 0.5ul Exo- are added
I enzymes；2.37 DEG C of incubation 15min, 80 DEG C of incubation 15min, inactivate unnecessary enzyme.Product after digestion processing is directly used in follow-up
ASPE primer extension reactions.
4th, the primer extension reaction (ASPE) of site-specific
Primer extension reaction is carried out using the ASPE primers of above-mentioned design, mixes biotin labeling during the reaction
DCTP, so that multiple biotin labeling in product band after reaction.
The ASPE primer working solutions of mixing are prepared first：The corresponding wild type of cls gene to be checked and saltant type ASPE are taken respectively
Primer stores liquid 10ul in 1.5ml microcentrifugal tubes, adds 10mmol/L Tris Buffer and mends to 200ul, is uniformly mixed
As ASPE mix primers working solution.The system of ASPE reactions is as follows：
| ASPE reaction systems | Often react (μ l) |
| NF water | 1.55 |
| NRAS extension primer mixed liquors | 5 |
| 10 × PCR Buffer (are free of Mg2+) | 2 |
| 25mM MgCl2 | 1 |
| Biotin-dCTP(400uM) | 0.25 |
| TspDNA polymerase | 0.2 |
| EXO/SAP products | 10 |
| Cumulative volume | 20 |
Response procedures are：96℃2min；94 DEG C of 30s, 54 DEG C of 1min, 72 DEG C of 2min, 30 circulations；4 DEG C save backup.
5th, hybridization reaction
It is 1. every kind of per the coated magnetic bead (as described in Example 1) of corresponding 21 kinds of group selection according to the ASPE primers of design
Magnetic bead concentration is 500/reaction, is prepared into magnetic bead mixed liquor
2. magnetic bead mixed liquor vortex 30s is mixed；
3. the above-mentioned magnetic bead mixed liquors of 45ul are taken in the 96 corresponding holes of hole filter plate
4. the ASPE reaction solutions of 5ul are taken to be mixed in corresponding hole with liquid-transfering gun
5 encase 96 orifice plates with masking foil is incubated hybridization with lucifuge, 95 DEG C of 60s, 37 DEG C of 15min；
96 orifice plates after 6 hybridization, which are placed on magnetic board, stands 1min, and liquid in reacting hole is outwelled in upset, 96 holes during this
Plate is close to magnetic board
7 are resuspended in magnetic bead in 1 × Tm hybridization buffers of 75ul；
8. 96 orifice plates, which are placed on magnetic board, stands 2min, liquid in reacting hole is outwelled in upset, and 96 orifice plates are close to during this
Magnetic board
9. 96 orifice plates are removed from magnetic board, 1 × Tm hybridization buffers that 75ul is added in reacting hole suspend magnetic again
Pearl, adds the streptavidin-phycoerythrin (SA-PE) that 15ul concentration is 10ug/ml；
12. 37 DEG C of incubation 15min, are detected on Luminex/MAGPIX instruments.
6th, result detection and data analysis
Product is detected by Luminex serial analysises instrument after reaction.It is more than 100 with saltant type fluorescent value (MFI)
Cut-off values, when the MFI values of saltant type detection are more than 100, judge that the sample there are the mutation type, otherwise judges the sample
This is corresponding wild type.
Cross reacting rate is detected the present invention carries out gene to the ASPE primers of the NRAS detection in Gene Mutation of design,
Experimental result shows (be shown in Table four and table five)；It is directed to the ASPE of special extension NRAS genotype (wild type and each mutation type)
Primer can specific amplification its corresponding detection type, amplification efficiency is high.And non-spy cannot be carried out for other mutation types
Opposite sex extension, even if there is the non-specific amplification of part, intergenic intersection amplified reaction rate is below 3%, therefore in the research
The specificity of ASPE primers is very high, and testing result is relatively reliable.
The present embodiment is analyzed for 18 samples, the results showed that (be shown in Table six and table seven)：18 parts of samples of detection
NRAS genotype call results and the sequencing result rate of coincideing reach 100%.It can be seen that NRAS gene mutations provided by the present invention
Detection liquid-phase chip can detect the mutation type of NRAS genes exactly, and result is reliable and stable.
Four ASPE primer cross reaction MFI values of table
Five ASPE primer cross reacting rates of table
Six NRAS of table detects sample results
Seven sample NRAS genotype results of table
| Sample label | Liquid-phase chip testing result | Sequencing result |
| 1 | A182G | A182G |
| 2 | Wild type | Wild type |
| 3 | G34C | G34C |
| 4 | Wild type | Wild type |
| 5 | Wild type | Wild type |
| 6 | G34A | G34A |
| 7 | Wild type | Wild type |
| 8 | Wild type | Wild type |
| 9 | Wild type | Wild type |
| 10 | Wild type | Wild type |
| 11 | Wild type | Wild type |
| 12 | Wild type | Wild type |
| 13 | Wild type | Wild type |
| 14 | Wild type | Wild type |
| 15 | Wild type | Wild type |
| 16 | Wild type | Wild type |
| 17 | Wild type | Wild type |
| 18 | Wild type | Wild type |
The selection of embodiment 3NRAS genetic polymorphism detections specific primer and probe sequence
One：The preparation (ASPE extension primers the are screened and screening of tag sequences) of liquid-phase chip
By taking 12 codon common gene mutations site G34A on 2 exons of NRAS gene mutations as an example, separately design
Specific primer sequence, using the complementary series forward or backwards of target sequence where the mutational site as template, is directed to respectively
Preferable specificity is drawn in wild type and saltant type the design specific probe sequence, including the embodiment of the present invention 1 in these sites
Thing and probe sequence and 2 (or more than 2) alternative specific primer and probe sequence, as shown in table 8,9.
Table 8G12S sites primer sequence filtered list
Table 9G12S locus specificities probe screens sequence table
Two：Sample survey
Using the liquid-phase chip of above-mentioned preparation, according to the detection process and method described in case study on implementation 2, to sample 19-
No. 40 samples are detected, and testing result is as follows：
Table 10：The primer sequence the selection result analysis of G12S sites
According to experimental result it can be seen that G12S site mutation type ASPE extension primers-SEQ ID NO.22 and wild type
ASPE extension primer SEQ ID NO.40 extend efficiency highest.
Table 11：The probe the selection result analysis of G12S sites
According to experimental result it can be seen that TAG sequence labels SEQ ID NO.1 and SEQ ID NO.2 are respectively as G12S
The tag sequence marks of point mutation type ASPE extension primers-SEQ ID NO.22 and wild type ASPE extension primer SEQ ID NO.40
During label, best results.
To sum up, SEQ ID NO.22, SEQ ID NO.40 are selected as G12S site mutation type ASPE extension primers, SEQ
ID NO.1 and SEQ ID NO.2 are respectively as its TAG sequence label.Other site primers and mark are carried out using identical method
The selection of sequence is signed, specific experiment data are omitted.
The selection of embodiment 4-NRAS genetic polymorphism detections magnetic bead or microballoon
Liquid-phase chip technology utilizes fluorescence-encoded microballoon or magnetic bead covalent cross-linking anti-tag sequences, which can be with
The tag sequence-specifics that allele-specific extension primer 5 ' is held are complementary.ASPE primers are combined with measured target molecule
Specifically extension target gene, formation include the extension products of tag sequences afterwards, then are made by hybridization reaction above ASPE primers
Tag sequences are combined with the anti-tag sequence specifics on magnetic bead or microballoon, finally utilize instrument luminex (magnetic bead microballoons
Be read) or each sample of MAGPIX (be only capable of read bead signal) analyses fluorescent value, sentenced according to the height of fluorescent value reading
The catastrophe of target gene in disconnected sample.It is to pass through microballoon to be used for liquid-phase chip technology before and be used for NRAS detection in Gene Mutation
Carry out, the reading of data can only be carried out by luminex instrument.This research replaces microballoon with magnetic bead, miscellaneous using its magnetism increase
Board-washing process after reacting is handed over, fluorescence signal value greatly improves, so that the sensitivity of detection is further enhanced, signal-to-noise ratio
Enhancing, testing result is more accurately and reliably.
By taking 12 codon common gene mutations site G34A on 2 exons of NRAS gene mutations as an example, magnetic is used respectively
Pearl is used for the detection of NRAS gene G34A sites extension primer with microballoon coating anti-tag sequences, as shown in table 12.
12 G12S loci detection method filtered lists of table
Two：Sample survey
Using the liquid-phase chip of above-mentioned preparation, according to the detection process and method described in case study on implementation 2, to sample 41-
No. 60 samples are detected, and testing result is as follows：
It can be seen that the magnetic bead for being coated with anti-tag sequences is coated with than anti-tag sequence according to above experimental result
Microballoon detect that fluorescence signal value greatly improves so that detection sensitivity be further enhanced, signal-to-noise ratio enhancing,
Testing result is more accurately and reliably.The primer and probe that the present invention designs, is more suitable for the bag by the use of magnetic bead as anti-tag sequences
It is better by carrier.Using identical method, to NRAS genes, other mutation types are detected, and specific data are omitted, knot
Fruit equally shows that magnetic bead is better as anti-tag coating carrier senses.
Each technical characteristic of the embodiment can be combined arbitrarily, to make description succinct, not to above-described embodiment
In each technical characteristic it is all possible combination be all described, as long as however, lance is not present in the combination of these technical characteristics
Shield, is all considered to be the scope of this specification record.
Embodiment described above only expresses the several embodiments of the present invention, its description is more specific and detailed, but simultaneously
Cannot therefore it be construed as limiting the scope of the patent.It should be pointed out that come for those of ordinary skill in the art
Say, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention
Scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Sequence table
<110>Surexam Biotechnology Co., Ltd.
<120>NRAS mutation detection kits and extension primer based on Luminex
<160> 73
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
tcaattactt cactttaatc cttt 24
<210> 2
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
aaacaaactt cacatctcaa taat 24
<210> 3
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
taattataca tctcatcttc taca 24
<210> 4
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
tcaatcatct ttatacttca caat 24
<210> 5
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
ctttttcaat cactttcaat tcat 24
<210> 6
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
cttttcaatt acttcaaatc ttca 24
<210> 7
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
aatctacaaa tccaataatc tcat 24
<210> 8
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
caataaacta tacttcttca ctaa 24
<210> 9
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
ttcataacta caatacatca tcat 24
<210> 10
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 10
tcaatcataa tctcataatc caat 24
<210> 11
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 11
aatccttttt actcaattca atca 24
<210> 12
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 12
cttttcatca ataatcttac cttt 24
<210> 13
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 13
tcatttcaat caatcatcaa caat 24
<210> 14
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 14
ttactcaaaa tctacacttt ttca 24
<210> 15
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 15
caatatcatc atctttatca ttac 24
<210> 16
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 16
ctacatattc aaattactac ttac 24
<210> 17
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 17
aaactaacat caatacttac atca 24
<210> 18
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 18
atacttcatt cattcatcaa ttca 24
<210> 19
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 19
cttctcatta acttacttca taat 24
<210> 20
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 20
ctacaaacaa acaaacatta tcaa 24
<210> 21
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 21
tacactttct ttctttcttt cttt 24
<210> 22
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 22
aaactggtgg tggttggagc aa 22
<210> 23
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 23
aaactggtgg tggttggagc at 22
<210> 24
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 24
aactggtggt ggttggagca c 21
<210> 25
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 25
actggtggtg gttggagcag a 21
<210> 26
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 26
ctggtggtgg ttggagcagc 20
<210> 27
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 27
actggtggtg gttggagcag t 21
<210> 28
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 28
ggtggtggtt ggagcaggtc 20
<210> 29
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 29
tggtggtggt tggagcaggt t 21
<210> 30
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 30
ggtggtggtt ggagcaggtg a 21
<210> 31
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 31
ggtggtggtt ggagcaggtg t 21
<210> 32
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 32
gtggtggttg gagcaggtgc 20
<210> 33
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 33
acatactgga tacagctgga a 21
<210> 34
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 34
catactggat acagctggac c 21
<210> 35
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 35
acatactgga tacagctgga ct 22
<210> 36
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 36
catactggat acagctggac g 21
<210> 37
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 37
catactggat acagctggac at 22
<210> 38
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 38
ttccattcat tgaaacctca ga 22
<210> 39
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 39
ttccattcat tgaaacctca gt 22
<210> 40
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 40
caggtggtgt tgggaaaagc g 21
<210> 41
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 41
atactggata cagctggaca a 21
<210> 42
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 42
aaaggattaa agtgaagtaa ttga 24
<210> 43
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 43
attattgaga tgtgaagttt gttt 24
<210> 44
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 44
tgtagaagat gagatgtata atta 24
<210> 45
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 45
attgtgaagt ataaagatga ttga 24
<210> 46
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 46
atgaattgaa agtgattgaa aaag 24
<210> 47
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 47
tgaagatttg aagtaattga aaag 24
<210> 48
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 48
atgagattat tggatttgta gatt 24
<210> 49
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 49
ttagtgaaga agtatagttt attg 24
<210> 50
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 50
atgatgatgt attgtagtta tgaa 24
<210> 51
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 51
attggattat gagattatga ttga 24
<210> 52
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 52
tgattgaatt gagtaaaaag gatt 24
<210> 53
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 53
aaaggtaaga ttattgatga aaag 24
<210> 54
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 54
attgttgatg attgattgaa atga 24
<210> 55
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 55
tgaaaaagtg tagattttga gtaa 24
<210> 56
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 56
gtaatgataa agatgatgat attg 24
<210> 57
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 57
gtaagtagta atttgaatat gtag 24
<210> 58
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 58
tgatgtaagt attgatgtta gttt 24
<210> 59
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 59
tgaattgatg aatgaatgaa gtat 24
<210> 60
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 60
attatgaagt aagttaatga gaag 24
<210> 61
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 61
ttgataatgt ttgtttgttt gtag 24
<210> 62
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 62
aaagaaagaa agaaagaaag tgta 24
<210> 63
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 63
ggctcgccaa ttaaccctga 20
<210> 64
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 64
tcacctctat ggtgggatca tat 23
<210> 65
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 65
ttgaacttcc ctccctccct g 21
<210> 66
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 66
tcgcctgtcc tcatgtattg g 21
<210> 67
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 67
tgccaacaag gacagttgat a 21
<210> 68
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 68
caaatgctga aagctgtacc atac 24
<210> 69
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 69
caaactggtg gtggttggag caa 23
<210> 70
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 70
acaaactggt ggtggttgga gcaa 24
<210> 71
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 71
agcaggtggt gttgggaaaa gcg 23
<210> 72
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 72
gagcaggtgg tgttgggaaa agcg 24
<210> 73
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 73
ttccattcat tgaaacctca gc 22
Claims (7)
1. a kind of NRAS gene mutation detection liquid-phase chips kit, it is characterized in that, include：
(A) is for No. 12 codons on 2 exons of NRAS genes, No. 13 codons, the 61st on 3 exons
18 mutator types in No. 146 codons and wild-type genotype on codon, 4 exons separately design ASPE and prolong
Extend thing, and every kind of ASPE extension primers are made of the tag sequences and 3 ' ends at 5 ' ends for the specific primer sequence in mutational site,
The specific primer sequence of mutator type is selected from following at least one：For the SEQ ID NO.22 in G34A sites,
For the SEQ ID NO.23 in G34T sites, for the SEQ ID NO.24 in G34C sites, for the SEQ ID in G35A sites
NO.25, for the SEQ ID NO.26 in G35C sites, for the SEQ ID NO.27 in G35T sites, for the SEQ in G37C sites
ID NO.28, for the SEQ ID NO.29 in G37T sites, for the SEQ ID NO.30 in G38A sites, for G38T sites
SEQ ID NO.31, for the SEQ ID NO.32 in G38C sites, for the SEQ ID NO.33 in C181A sites, for A182C
The SEQ ID NO.34 in site, for the SEQ ID NO.35 in A182T sites, for the SEQ ID NO.36 in A182G sites, pin
To the SEQ ID NO.37 in A183T sites, for the SEQ ID NO.38 in G436A sites, for the SEQ ID in G437T sites
NO.39；
The ASPE extension primers of wild-type genotype are selected from corresponding following at least one：For the SEQ ID of 2 exons
NO.40, for 3 exon SEQ ID NO.41, for 4 exon SEQ ID NO.73；
(B) has the magnetic bead that anti-tag sequences are coated, there are different colours to encode, and the anti-tag sequences connect with magnetic bead
Connect centre and be additionally provided with spacer sequence；The anti-tag sequences are selected from SEQ ID NO.42~SEQ ID NO.62, and described
Anti-tag sequences can be matched correspondingly with tag sequences complete complementary selected in (A)；
(C) is used to amplify the primer for needing target sequence detect, with corresponding mutational site.
2. NRAS gene mutation detection liquid-phase chips kit according to claim 1, it is characterized in that, the amplimer
For selected from following at least a pair of：For SEQ ID NO.63 and SEQ the ID NO.64 of 2 exons, for 3 exon SEQ
ID NO.65 and SEQ ID NO.66, for 4 exon SEQ ID NO.67 and SEQ ID NO.68.
3. NRAS gene mutation detection liquid-phase chips kit according to claim 1, it is characterized in that, mutator type
The ASPE extension primers be selected from it is at least one of following：For G34A sites by SEQ ID NO.1 and SEQ ID
The sequence of NO.22 compositions, for the sequence being made of SEQ ID NO.2 and SEQ ID NO.23 in G34T sites, for G34C
The sequence being made of SEQ ID NO.3 and SEQ ID NO.24 in site, for G35A sites by SEQ ID NO.4 and SEQ
The sequence of ID NO.25 compositions, for the sequence being made of SEQ ID NO.5 and SEQ ID NO.26 in G35C sites, for
The sequence being made of SEQ ID NO.6 and SEQ ID NO.27 in G35T sites, for G37C sites by SEQ ID NO.7 and
The sequence of SEQ ID NO.28 compositions, for the sequence being made of SEQ ID NO.8 and SEQ ID NO.29 in G37T sites, pin
To the sequence being made of SEQ ID NO.9 and SEQ ID NO.30 in G38A sites, for G38T sites by SEQ ID
The sequence of NO.10 and SEQ ID NO.31 compositions, is made of for G38C sites SEQ ID NO.11 and SEQ ID NO.32
Sequence, for the sequence being made of SEQ ID NO.12 and SEQ ID NO.33 in C181A sites, for A182C sites
The sequence being made of SEQ ID NO.13 and SEQ ID NO.34, for A182T sites by SEQ ID NO.14 and SEQ ID
The sequence of NO.35 compositions, for the sequence being made of SEQ ID NO.15 and SEQ ID NO.36 in A182G sites, for
The sequence being made of SEQ ID NO.16 and SEQ ID NO.37 in A183T sites, for G436A sites by SEQ ID
The sequence of NO.17 and SEQ ID NO.38 compositions, is made of for G437T sites SEQ ID NO.18 and SEQ ID NO.39
Sequence；
Corresponding at least one of the ASPE extension primers described in wild-type genotype in following：For 2 exons by SEQ
The sequence of ID NO.19 and SEQ ID NO.40 compositions, for 3 exons, by SEQ ID NO.20 and SEQ ID NO.41 groups
Into sequence, for 4 exons, the sequence being made of SEQ ID NO.21 and SEQ ID NO.73.
4. according to claim 1-3 any one of them NRAS gene mutation detection liquid-phase chip kits, it is characterized in that, including
The corresponding ASPE extension primers in 18 mutational sites having the right described in requirement 1, the magnetic bead and the amplimer.
5. according to claim 1-3 any one of them NRAS gene mutation detection liquid-phase chip kits, it is characterized in that, it is described
Spacerarm is 5-10 T.
6. for the ASPE extension primers of NRAS detection in Gene Mutation liquid, it is characterized in that, on 2 exons of NRAS genes
No. 12 codons, No. 13 codons, the 61st bit codon on 3 exons, in No. 146 codons on 4 exons
18 mutator types and wild-type genotype separately design ASPE extension primers, every kind of ASPE extension primers are by 5 ' ends
Tag sequences and 3 ' ends are formed for the specific primer sequence in mutational site, the specific primer sequence of mutator type
Column selection is from following at least one：For the SEQ ID NO.22 in G34A sites, for the SEQ ID NO.23 in G34T sites, for
The SEQ ID NO.24 in G34C sites, for the SEQ ID NO.25 in G35A sites, for the SEQ ID NO.26 in G35C sites,
For the SEQ ID NO.27 in G35T sites, for the SEQ ID NO.28 in G37C sites, for the SEQ ID in G37T sites
NO.29, for the SEQ ID NO.30 in G38A sites, for the SEQ ID NO.31 in G38T sites, for the SEQ in G38C sites
ID NO.32, for the SEQ ID NO.33 in C181A sites, for the SEQ ID NO.34 in A182C sites, for A182T
The SEQ ID NO.35 of point, for the SEQ ID NO.36 in A182G sites, for the SEQ ID NO.37 in A183T sites, for
The SEQ ID NO.38 in G436A sites, for the SEQ ID NO.39 in G437T sites；
The ASPE extension primers of wild-type genotype are selected from corresponding following at least one：For the SEQ ID of 2 exons
NO.40, for 3 exon SEQ ID NO.41, for 4 exon SEQ ID NO.73；
The tag sequences are selected from：SEQ ID NO.1—SEQ ID NO.21.
7. the ASPE extension primers according to claim 6 for NRAS detection in Gene Mutation liquid, it is characterized in that, it is mutated base
Because the ASPE extension primers of type are selected from least one of following：For G34A sites by SEQ ID NO.1 and SEQ
The sequence of ID NO.22 compositions, for the sequence being made of SEQ ID NO.2 and SEQ ID NO.23 in G34T sites, for
The sequence being made of SEQ ID NO.3 and SEQ ID NO.24 in G34C sites, for G35A sites by SEQ ID NO.4 and
The sequence of SEQ ID NO.25 compositions, for the sequence being made of SEQ ID NO.5 and SEQ ID NO.26 in G35C sites, pin
To the sequence being made of SEQ ID NO.6 and SEQ ID NO.27 in G35T sites, for G37C sites by SEQ ID NO.7
With the sequence of SEQ ID NO.28 compositions, for the sequence being made of SEQ ID NO.8 and SEQ ID NO.29 in G37T sites,
For the sequence being made of SEQ ID NO.9 and SEQ ID NO.30 in G38A sites, for G38T sites by SEQ ID
The sequence of NO.10 and SEQ ID NO.31 compositions, is made of for G38C sites SEQ ID NO.11 and SEQ ID NO.32
Sequence, for the sequence being made of SEQ ID NO.12 and SEQ ID NO.33 in C181A sites, for A182C sites
The sequence being made of SEQ ID NO.13 and SEQ ID NO.34, for A182T sites by SEQ ID NO.14 and SEQ ID
The sequence of NO.35 compositions, for the sequence being made of SEQ ID NO.15 and SEQ ID NO.36 in A182G sites, for
The sequence being made of SEQ ID NO.16 and SEQ ID NO.37 in A183T sites, for G436A sites by SEQ ID
The sequence of NO.17 and SEQ ID NO.38 compositions, is made of for G437T sites SEQ ID NO.18 and SEQ ID NO.39
Sequence；
Corresponding at least one of the ASPE extension primers described in wild-type genotype in following：For 2 exons by SEQ
The sequence of ID NO.19 and SEQ ID NO.40 compositions, for 3 exons, by SEQ ID NO.20 and SEQ ID NO.41 groups
Into sequence, for 4 exons, the sequence being made of SEQ ID NO.21 and SEQ ID NO.73.
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| CN112176062A (en) * | 2020-10-13 | 2021-01-05 | 苏州中科先进技术研究院有限公司 | Nucleic acid composition for detecting NRAS gene mutation and kit thereof |
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