CN106676105A - Primers capable of simultaneously acquiring and detecting multiple target area sequences, kit and method - Google Patents
Primers capable of simultaneously acquiring and detecting multiple target area sequences, kit and method Download PDFInfo
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Abstract
The invention provides multiple PCR primers capable of amplifying and/or detecting target gene areas, such as amplifying and detecting mutant sites of EGFR, KRAS and/or BRAF genes. By using the multiple PCR primers, the mutant sites of EGFR/KRAS/BRAF genes can be efficiently detected in one time, and a plurality of exons on the EGFR, KRAS and/or BRAF genes can be simultaneously covered.
Description
Related application
The application is to be on November 4th, 2015 applying date, and entitled " one kind is based on sequencing technologies detection EGFR/ of future generation
The Chinese patent application 201510740906.1 of the multiple PCR primer and method of KRAS/BRAF gene mutation sites and application "
Division.
Technical field
The invention belongs to biology field, it is more particularly to a kind of based on sequencing technologies detection EGFR of future generation,
The multiple PCR primer and method and application in KRAS and/or BRAF gene mutation site.
Background technology
EGF-R ELISA (epidermal growth factor receptor, EGFR) is positioned at No. 7 dyeing
The expression product of the proto-oncogene C-erbB-1 (HER-1) on body galianconism, is 4 in Epidermal Growth Factor Receptor Family (HER)
One of individual member, HER families play important adjustment effect in cellular physiological processes.The mutation of EGFR tyrosine kinase domains
18~21 exons are occurred mainly in, wherein the mutation of 19 and 21 exons accounts for the 90% of total mutation.EGFR signal transductions
Exception is the reason for causing kinds of tumors to occur.Research shows that EGFR has expression, such as colorectal cancer, breast in kinds of tumors
Adenocarcinoma, cancer of pancreas, carcinoma of prostate and nonsmall-cell lung cancer etc..
K-ras genes are a kind of proto-oncogenes, are about 35kb, on No. 12 the short arm of a chromosome, be ras gene families into
One of member, encoded K RAS albumen.The common mutational site of K-ras genes is located at No. 12 codons of 2 exons and No. 13 close
On numeral, the 6l codons of 3 exons, wherein there is 7 mutantional hotspots：G12C、G12R、G12S、G12V、G12D、G12A、
G13V/D, accounts for more than the 98% of the always mutation of K-ras genes.Research shows somatic cell K-ras gene mutation with various human malignants
Tumor, such as pulmonary carcinoma, leukemia, mucoprotein adenocarcinoma, cancer of pancreas, colorectal cancer are relevant, and sexual cell K-ras gene mutation with exert
Southern syndrome and heart-face-skin (cardio-facio-cutaneous, CFC) syndrome are related.
BRAF gene is a kind of proto-oncogene, is positioned on No. 7 chromosomes, encodes serine/threonine specificity kinase.About
90% BRAF gene mutation occurs on 1799 nucleotide of exon 15, and T sports A, causes L-Valine by glutamic acid
Replace (V600E).BRAF gene mutation melanoma, colorectal cancer, glioma, sarcoma, ovarian cancer, breast carcinoma and
Incidence rate in the tumor cell lines such as pulmonary carcinoma is followed successively by 59%, 18%, 11%, 9%, 14%, 2%, 3%；Additionally, BRAF
Incidence rates of the V600E in thyroid papillary carcinoma is up to 35.8%.BRAF gene mutation is late period and recurrent colorectal cancer
One of most important prognostic indicator, the overall survival phase poor with patient is closely related.
The method for detecting these three gene mutation common at present has Sanger sequencing and fluorescence quantitative PCR method.
In Sanger sequencing, single pair of primer can detect in multiple mutation, but the different exons to multiple genes or same gene
Mutational site just needs to carry out amplification sequencing respectively, cumbersome, and sensitivity relatively low about 20%, and false negative rate is higher.Fluorescence
Although quantitative PCR method sensitivity is high, each pair primer can only detect a kind of mutation, and every kind of mutation need to individually set up a PCR
Reaction system, while detecting that the multiple mutational sites of multiple samples are cumbersome.Both approaches are all larger to the requirement of sample,
Be not suitable for while detecting multiple gene mutation sites.
The content of the invention
In consideration of it, the invention provides a kind of multiple PCR primer and application.
In a first aspect, a kind of the invention provides multiple PCR primer.The multiple PCR primer is made up of following primer pair：
SEQ ID NO:1 and SEQ ID NO:Primer pair shown in 2, SEQ ID NO:5 and SEQ ID NO:Primer pair shown in 6, SEQ ID
NO:9 and SEQ ID NO:Primer pair shown in 10, SEQ ID NO:13 and SEQ ID NO:Primer pair shown in 14, SEQ ID NO:
15 and SEQ ID NO:Primer pair shown in 16 and SEQ ID NO:19 and SEQ ID NO:Primer pair shown in 20.
Inventor is based on many secondary designs of target sequence feature, and repeatedly composite test screening, it is determined that this group is multiple
PCR primer, using the multiple PCR primer, disposably efficient amplification can obtain the sequence of the multiple exons on EGFR, then
Can be used in detecting the gene mutation site on the multiple exons on EGFR.SEQ ID NO therein:1 and SEQ ID NO:2
Primer pair can not be realized the region of the exons 18 of EGFR gene in which be affected in same reaction system by other primer pairs
At least one of amplification, SEQ ID NO:5 and SEQ ID NO:6 primer pairs can not drawn by other in same reaction system
Thing to affecting realizes at least one of amplification in the region of the exons 19 of EGFR gene, SEQ ID NO:9 and SEQ ID
NO:10 primer pairs and SEQ ID NO:13 and SEQ ID NO:14 primer pairs can not drawn by other in same reaction system
Thing to affecting realizes at least one of amplification in the region of the extron 20 of EGFR gene, SEQ ID NO:15 and SEQ ID
NO:16 primer pairs and SEQ ID NO:19 and SEQ ID NO:20 primer pairs can not drawn by other in same reaction system
Thing to affecting realizes at least one of amplification in the region of the exon 21 of EGFR gene.
Embodiments in accordance with the present invention, above-mentioned multiple PCR primer can further include following additional technical feature extremely
It is one of few：
Embodiments in accordance with the present invention, the multiple PCR primer also includes at least one pair of in following primer pair：SEQ ID
NO:3 and SEQ ID NO:Primer pair shown in 4, SEQ ID NO:7 and SEQ ID NO:Primer pair shown in 8, SEQ ID NO:11 Hes
SEQ ID NO:Primer pair shown in 12, SEQ ID NO:17 and SEQ ID NO:Primer pair shown in 18, SEQ ID NO:21 and SEQ
ID NO:Primer pair shown in 22, SEQ ID NO:23 and SEQ ID NO:Primer pair shown in 24, SEQ ID NO:25 and SEQ ID
NO:26 primer pairs and SEQ ID NO:27 and SEQ ID NO:Primer pair shown in 28.Embodiments in accordance with the present invention, this is multiple
PCR primer also includes two couples, three pairs, five pairs or whole eight pairs in primer pair listed above.Wherein, SEQ ID NO:3 Hes
SEQ ID NO:4 primer pairs by other primer pairs can not be realized the exon of EGFR gene in which be affected in same reaction system
At least one of amplification in 18 region, SEQ ID NO:7 and SEQ ID NO:8 primer pairs can be in same reaction system
At least one of amplification in the region of the exons 19 of EGFR gene, SEQ ID NO are not realized by other primer pairs with being affected:
11 and SEQ ID NO:12 primer pairs by other primer pairs can not be realized EGFR gene with being affected in same reaction system
At least one of amplification in the region of extron 20, SEQ ID NO:17 and SEQ ID NO:18 primer pairs can be same anti-
Answer the region of do not realized amplification EGFR gene in system by other primer pairs exon 21 with being affected, SEQ ID NO:21 Hes
SEQ ID NO:22 primer pairs by other primer pairs can not be realized the outer aobvious of KRAS genes in which be affected in same reaction system
At least one of amplification in the region of son 2, SEQ ID NO:23 and SEQ ID NO:24 primer pairs can be in same reactant
At least one of amplification in the region of the exon 2 of KRAS genes, SEQ ID are not realized in system by other primer pairs with being affected
NO:25 and SEQ ID NO:26 primer pairs by other primer pairs can not be realized BRAF gene with being affected in same reaction system
Exons 15 region at least one of amplification, SEQ ID NO:27 and SEQ ID NO:28 primer pairs can be same
At least one of amplification in the region of the exons 15 of BRAF gene is not realized in reaction system by other primer pairs with being affected.
Embodiments in accordance with the present invention, in the multiple PCR primer, at least including amplification EGFR, KRAS and BRAF gene
The primer pair of middle any two gene, the primer pair can be used in expanding at least one exon region of the gene.
If it will be understood by those skilled in the art that the multiplex PCR that many secondary designs are groped and test of many times screening determines draws
Thing group can be in same reaction system, while realize the amplification of target fragment without interfering with each other, any portion primer therein
Combination also can play respective function in same reaction system, i.e., independently realize the expansion in respective objects region simultaneously
Increase.
Embodiments in accordance with the present invention, the multiple PCR primer is SEQ ID NO:1~SEQ ID NO:Core shown in 28
The primer sets of nucleotide sequence composition.The disposable amplified production for obtaining can comprehensively cover the exons of EGFR 18~21, KRAS 2
Exon and the exon regions of BRAF 15.
If it will be understood by those skilled in the art that the multiplex PCR that many secondary designs are groped and test of many times screening determines draws
Thing group (such as, SEQ ID NO:1~SEQ ID NO:The primer sets of the nucleotide sequence composition shown in 28) obtain preferably
Expanding effect, generally, when design primer, the appropriate length by an arbitrary primer in multiple PCR primer group
Extend or 0~3 base of truncate, it is also possible to obtain good the Efficiency of Mutiplex PCR, should also include the scope of the present invention.
Second aspect, the invention provides above the multiple PCR primer in one aspect of the present invention or any embodiment exists
Obtain and/or detect EGFR, KRAS and/or the purposes in BRAF gene sequence.It is above-mentioned to one aspect of the present invention or arbitrary reality
The technical characteristic and the description of effect, the purposes of equally applicable this aspect of the present invention of multiple PCR primer in example are applied, here is not
Repeat again.
The third aspect, the invention provides a kind of test kit.The test kit includes above one aspect of the present invention or arbitrary
Multiple PCR primer in embodiment.The technology of the above-mentioned multiple PCR primer in one aspect of the present invention or any embodiment is special
The description of effect of seeking peace, the test kit of equally applicable this aspect of the present invention, will not be described here.
Fourth aspect, the invention provides above the test kit in any embodiment is obtaining and/or detecting EGFR, KRAS
And/or the purposes in BRAF gene sequence.
5th aspect, the invention provides a kind of method of amplification gene sequence target area.Enforcement of the invention
Example, methods described includes carrying out the amplification using foregoing multiple PCR primer.
Embodiments in accordance with the present invention, said method can further include at least one following additional technical feature：
According to a particular embodiment of the invention, in the system of the multi-PRC reaction, each primer equimolar mixing.
Still another embodiment of the invention, in the system of the multi-PRC reaction, template amount is 50ng~1 μ g/
50 μ l systems.
Still another embodiment of the invention, the program of the multi-PRC reaction is：
Said procedure is specially：95 DEG C of denaturations 15min, 95 DEG C of degeneration 15s, 60 DEG C of annealing 2min, 72 DEG C of extension 3min,
Circulation 30~40 times (preferably 35 times), after last 72 DEG C 10min is extended.
Specific example of the invention, after the multi-PRC reaction terminates, electrophoresis, rubber tapping is reclaimed fragment length and is
The DNA fragmentation of 150bp.
6th aspect, the invention provides a kind of EGFR, KRAS and/or the method for BRAF gene detection.According to the present invention
Embodiment, methods described includes：(1) at least a portion nucleic acid treated using foregoing method in sample is expanded
Increase, obtain amplified production；(2) amplified production is analyzed, to obtain EGFR, KRAS and/or BRAF gene testing result.
The method of embodiments in accordance with the present invention, above-mentioned EGFR, KRAS and/or BRAF gene detection can also be wrapped further
Include at least one following additional technical feature：
Embodiments in accordance with the present invention, the nucleic acid is DNA and/or RNA.
Embodiments in accordance with the present invention, the RNA is that human peripheral blood single nucleus cell is extracted (preferably using RNA test kits)
The total serum IgE of acquisition.
Embodiments in accordance with the present invention, the nucleic acid is RNA, and step (1) includes：(1-1) multiple PCR primer is utilized
In upstream or downstream primer by the RNA reverse transcriptions be cDNA；And (1-2) is using corresponding in the multiple PCR primer
Downstream or forward primer are expanded to the cDNA, obtain amplified production.According to a particular embodiment of the invention, when the core
Acid is RNA, and step (1) includes：First synthesize cDNA by reverse transcription primer of downstream primer sets；Then the cDNA with synthesis is as mould
Plate, adds forward primer group, carries out multiplex PCR, expands cDNA, obtains multiple PCR products.
Still another embodiment of the invention, when the nucleic acid is RNA, step (1) includes：First with upstream primer sets
Synthesize cDNA for reverse transcription primer；Then the cDNA with synthesis adds downstream primer group as template, carries out multiplex PCR, expands
CDNA, obtains multiple PCR products.
Embodiments in accordance with the present invention, step (2) includes：(2-1) amplified production is sequenced；And (2-2)
By sequencing result respectively with EFGR, KRAS and/or BRAF gene wild-type sequence is compared, to determine EFGR, KRAS and/or BRAF
Gene mutation site.
As described in the present invention, described " EGFR, KRAS and/or BRAF gene mutation site " or " EGFR/KRAS/BRAF
Gene mutation site " represents one or more in tri- genes of EGFR, KRAS, BRAF.
Eighth aspect, the present invention proposes a kind of method of detection cancer, embodiments in accordance with the present invention, methods described bag
Include：The method detected using foregoing EGFR, KRAS and/or BRAF gene carries out EFGR, KRAS and/or BRAF gene inspection
Survey, obtain testing result, the testing sample is from person under inspection；And according to testing result, that assesses person under inspection suffers from cancer risk.
Embodiments in accordance with the present invention, the method for above-mentioned detection cancer can further include following additional technical feature
At least one：
Embodiments in accordance with the present invention, the testing result includes at least one of following mutation：EGFR c.2156G>
A、EGFR c.2235_2249del15、EGFR c.2240_2257del18、EGFR c.2369C>T、EGFR c.2573T>G、
EGFR c.2582T>A、EGFR c.34G>T、KRAS c.34G>T、KRAS c.34G>A、KRAS c.34G>C、KRAS c.35G
>T、KRAS c.35G>A、KRAS c.35G>C、KRAS c.38G>A and BRAF are c.1799T>A, it indicates that person under inspection suffers from cancer
Disease.Above-mentioned mutation is to mark position of the site on reference to cDNA according to nomenclatures such as HGVS to represent, a mutation position
Point can also have other representations, the such as naming method of GenBank snp databases, be the SNP site table started with " rs "
Show mode, rs671 is with ALDH2 genes c.1510G>A (G1510A) represents same site.Those of ordinary skill in the art know
Road, the position for adopting other naming methods such as with the mutation on reference to gDNA is marking the mutation referred to as the present invention
Fall within the scope of the invention.
Embodiments in accordance with the present invention, the cancer is included selected from colorectal cancer, breast carcinoma, cancer of pancreas, carcinoma of prostate, non-
Small cell lung cancer, leukemia, mucoprotein adenocarcinoma, melanoma, glioma, sarcoma, ovarian cancer and breast carcinoma at least it
One.
The multiplex PCR that EGFR/KRAS/BRAF gene mutation sites are detected based on sequencing technologies of future generation that the present invention is provided
Primer and method have the beneficial effect that：The invention provides the multiplex PCR in detection EGFR, KRAS and BRAF gene mutation site draws
Thing, contains the primer pair of one or more exon regions for expanding these three genes respectively, can cover EGFR 18~21
Exon, the exons of KRAS 2 and the exons of BRAF 15, include the Primary mutations of EGFR, KRAS and BRAF gene
Site.Designed target sequence is effectively expanded in each DNA sample, in designed KRAS, EGFR and
Mutation can be really detected in each exon of BRAF gene, so as to reflect that it is preferable that provided detection scheme has
Specificity and the suitability.
The additional aspect and advantage of the present invention will be set forth in part in the description, and partly will become from the following description
Obtain substantially, or recognized by the practice of the present invention.
Description of the drawings
The above-mentioned and/or additional aspect and advantage of the present invention will become from the description with reference to accompanying drawings below to embodiment
It is substantially and easy to understand, wherein：
Fig. 1 is the agarose gel electrophoresis figure of independent primer PCR provided in an embodiment of the present invention；
Fig. 2 is the agarose gel electrophoresis figure of multiplex PCR provided in an embodiment of the present invention；And
Fig. 3 is the bioinformatic analysis result of sequencing depth provided in an embodiment of the present invention.
Specific embodiment
Embodiments of the invention are described below in detail, the example of the embodiment is shown in the drawings, wherein from start to finish
Same or similar label represents same or similar element or the element with same or like function.Below with reference to attached
The embodiment of figure description is exemplary, is only used for explaining the present invention, and is not considered as limiting the invention.
Material and reagent explanation：
Cancer (such as pulmonary carcinoma, colorectal cancer) patient：From Shenzhen Sixth Man people hospital, patient's informed consent.Non- spy
Different explanation, the reagent that the embodiment of the present invention is adopted is commercial goods, and the data base that the embodiment of the present invention is adopted is disclosed
Online database.
Specifically, primer of the present invention is as shown in table 1.
Table 1：Multiple PCR primer sequence
Design primer：Primer dimer and stem ring mispairing are carried out point using Oligo 7.0 and MFEprimer-2.0
Analysis, in the design of the exon two ends comprising mutational site primer, the sequence average length of amplification is in 150bp or so, and 14 pairs are drawn
The annealing temperature of thing is basically identical.
The primer sets that the present embodiment is provided cover the exons of EGFR 18~21, the exons of KRAS 2 and BRAF 15
Exon gene mutation site.Because the sequence variation of very little will cause primer expanding effect to significantly reduce, inventor's difference
Multigroup multiple PCR primer group, after screening through preliminary experiment, condensation products are devised for the different sections of different purpose regions
Fragment length and point mutation coverage, the present invention have chosen the optimal primer sets of expanding effect, as shown in Table 1.
The independent PCR results of 1 each pair of primer of table are as shown in Figure 1.Wherein, 14 pairs of primers carry out expanding checking respectively in table 1
PCR system and PCR parameters are as follows：
Reaction system is as shown in table 2：With reference to QIAGEN company Multiplex PCR kit (article No.s：206143) configure.
Table 2：
| Multiplex PCR buffer (Multiplex Buffer, 2 ×) | 25μl |
| Q solvents (Q solution, 5 ×) | 10μl |
| Primer | 5μl |
| DNA | 10μl |
| Total | 50μl |
Loop parameter：
Embodiment 1
The embodiment of the present invention 1 provides a kind of preparation method of DNA sample to be measured, comprises the steps：
The tissue comprising cancerous cell is obtained from hospital, base is extracted using QIAamp DNA Mini Kit (51304) test kits
Because of a group DNA, and the concentration and purity for determining DNA with Nanodrop2000 (Thermo), then preserve genomic DNA.
Embodiment 2
The embodiment of the present invention 2 provides a kind of multiplex PCR using detection EGFR/KRAS/BRAF gene mutation sites and draws
The method that thing builds EGFR/KRAS/BRAF gene mutation sequencing libraries, comprises the steps：
1st, multiplex PCR：
SEQ ID NO are adopted with the gained genomic DNA of embodiment 1 to expand template:1~SEQ ID NO:14 pairs shown in 28
Primer pair, then using QIAGEN companies Multiplex PCR kit (article No.s：206143), two are configured by kit specification
Pipe multiplex PCR system, multiplex PCR system is as shown in table 3.
Table 3：Reaction system：
| Multiplex PCR buffer (Multiplex Buffer, 2 ×) | 25μl |
| Q solvents (Q solution, 5 ×) | 10μl |
| Primer | 5μl |
| DNA | 10μl |
| Total | 50μl |
Each primer is divided to two groups to carry out equimolar mixing, and total primer concentration is 10 micromoles.First group of primer includes：Primer is compiled
Numbers 1,2,5,6,9,10,13,14,15,16,21,22,25 and 26；Second group of primer includes：Primer numbers 3,4,7,8,11,12,
17th, 18,19,20,23,24,27 and 28.Template amount can be adjusted, and 200ng is adopted in the present embodiment.
Again by the condition setting PCR instrument device program of following multiplex PCRs, multiplex PCR is carried out：
After PCR terminates, 4 DEG C preserve PCR primer and electrophoresis detection, and the purpose fragment of about 150bp or so is cut under ultraviolet.
The PCR primer of first group of primer and second group of primer is combined into recovery, 32uL products after purification, glue reclaim step is obtained
Using QIAGEN companies QIAquick gel purification kits, routinely laboratory operation is carried out.
2nd, tailing：
PCR primer after purification is taken, in the end of product 3 ' A tails, configuration scheme (wherein, Klenow as shown in table 4 are added
Exo- is purchased from NEB, article No.：M0212)：
Table 4：
| 10×NEB2 BUFFER | 5μl |
| dATP(1mM) | 10μl |
| Klenow exo- | 3μl |
| DNA | 32μl |
| Total | 50μl |
30min at the system is placed in into 37 DEG C.Add A tails using QIAGEN companies QIAquick gel purification kit purification
PCR primer.
3rd, joint is added：
DNA two ends plus sequencing joint, configuration scheme as shown in table 5 (wherein, Quick ligase are purchased from NEB,
M2200L)。
Table 5：
| 5×quick ligase buffer | 10μl |
| Adaptor | 1μl |
| Quick ligase | 5μl |
| Plus the PCR primer of A tails | 25μl |
| H2O | 9μl |
| Total | 50μl |
Wherein, the sequence SEQ ID NO of Adaptor：Shown in 29：
5’-/5Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTC/ideoxyU/
ACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3’(SEQ ID NO：29).
15min at subsequently the system being placed in into 20 DEG C.It is subsequently adding the USER of 3 μ L, 37 DEG C of placement 15min.Finally by
QIAGEN companies QIAquick glue reclaim kits connection products.
4th, the sequence label of sequencing is added on product, as shown in table 6 (concrete steps are high with reference to illumina for configuration scheme
Flux sequencing library builds description).
Table 6：
| 5×Q5 Solution buffer | 10μl |
| dNTP(10mM) | 1μl |
| P1 (P5 sequences+common sequences+joint sequence) | 1μl |
| Index (P7 sequences+sequence label Index+ joint sequences) | 1μl |
| Q5 enzymes | 0.5μl |
| Connection product after purification | 36.5ul |
| Total | 50μl |
The system of configuration is entered into performing PCR reaction by following programs：
After PCR terminates, the size of PCR primer rich segment is verified with 1.5% agarose gel electrophoresiies, choose 270bp's
Purpose fragment carries out cutting glue reclaim, and is purified to 30 μ L.
Shown in agarose gel electrophoresiies result Fig. 2.In fig. 2, S1~S4 (representing different samples respectively) swimming lane can be with
Be clearly visible has a band between 250bp-300bp, is the fragment (270bp, at arrow indication) for adding top connection, with theoretical phase
Symbol.
5th, the purified product for obtaining step 4 is directly sequenced with Miseq platforms.
Sequencing result is the data of fastq forms, and by bioinformatic analysis tri- bases of EGFR, BRAS and KRAF are obtained
Because of multi-mutant site situation.Sequencing depth is shown in Fig. 3.The present embodiment is sequenced using PE150 test kits, i.e. fragment two ends are respectively surveyed
Sequence 150bp.Depth is sequenced for bioinformatic analysis as shown in figure 3, abscissa is EGFR, BRAS and KRAF each pair primer obtains
Amplified production, vertical coordinate is sequencing depth.From the figure 3, it may be seen that covering the amplification of all primer pairs in the present embodiment sequencing result
Product, and be distributed average.
Effect example 1
With reference to the method for embodiment 2,100 pulmonary carcinoma cancer specimens (sample comprising cancerous cell) are carried out respectively multiple
PCR, plus A tails, plus joint, the sequence that tags, high-flux sequence and bioinformatic analysis, the results are as follows, such as the institute of table 7
Show：
Table 7：Cancer specimen gene mutation site is detected
As shown in table 7, in 100 cancer specimens of test, the Primary mutations site of three genes detects.It is special
Not it should be noted that the data analysiss through being sequenced to sample, it was demonstrated that designed target sequence is equal in each DNA sample
Effectively expanded, from mutation result in it can also be seen that, in each exon of designed KRAS, EGFR and BRAF gene
In detect mutation, so as to reflect that provided detection scheme has preferable specificity and the suitability.
In describing the invention, unless otherwise stated, " multiple " are meant that two or more.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " illustrative examples ",
The description of " example ", " specific example " or " some examples " etc. means to combine specific features, the knot that the embodiment or example are described
Structure, material or feature are contained at least one embodiment of the present invention or example.In this manual, to above-mentioned term
Schematic representation is not necessarily referring to identical embodiment or example.And, the specific features of description, structure, material or spy
Point can in an appropriate manner be combined in any one or more embodiments or example.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that：Not
These embodiments can be carried out with various changes, modification, replacement and modification in the case of the principle and objective that depart from the present invention, this
The scope of invention is limited by claim and its equivalent.
SEQUENCE LISTING
<110>Big desert gene biological Science and Technology Ltd. of Shenzhen
<120>Primer, test kit and the method for multiple target area sequences are obtained and detected simultaneously
<130> PI2015017_4
<160> 29
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> Artificial
<220>
<223>Multiple PCR primer（EGFR-18-No.1-F）
<400> 1
cccttgtctc tgtgttcttg 20
<210> 2
<211> 19
<212> DNA
<213> Artificial
<220>
<223>Multiple PCR primer（EGFR-18-No.1-R）
<400> 2
cttatacacc gtgccgaac 19
<210> 3
<211> 20
<212> DNA
<213> Artificial
<220>
<223>Multiple PCR primer（EGFR-18-No.2-F）
<400> 3
gagaagctcc caaccaagct 20
<210> 4
<211> 18
<212> DNA
<213> Artificial
<220>
<223>Multiple PCR primer（EGFR-18-No.2-R）
<400> 4
agaccatgag aggccctg 18
<210> 5
<211> 19
<212> DNA
<213> Artificial
<220>
<223>Multiple PCR primer（EGFR-19-No.3-F）
<400> 5
gcagcatgtg gcaccatct 19
<210> 6
<211> 20
<212> DNA
<213> Artificial
<220>
<223>Multiple PCR primer（EGFR-19-No.3-R）
<400> 6
ttccttgttg gctttcggag 20
<210> 7
<211> 20
<212> DNA
<213> Artificial
<220>
<223>Multiple PCR primer（EGFR-19-No.4-F）
<400> 7
tctctctgtc atagggactc 20
<210> 8
<211> 21
<212> DNA
<213> Artificial
<220>
<223>Multiple PCR primer（EGFR-19-No.4-R）
<400> 8
cacagcaaag cagaaactca c 21
<210> 9
<211> 20
<212> DNA
<213> Artificial
<220>
<223>Multiple PCR primer（EGFR-20-No.1-F）
<400> 9
atgcgaagcc acactgacgt 20
<210> 10
<211> 18
<212> DNA
<213> Artificial
<220>
<223>Multiple PCR primer（EGFR-20-No.1-R）
<400> 10
gatgagctgc acggtgga 18
<210> 11
<211> 18
<212> DNA
<213> Artificial
<220>
<223>Multiple PCR primer（EGFR-20-No.2-F）
<400> 11
ctccctccag gaagccta 18
<210> 12
<211> 21
<212> DNA
<213> Artificial
<220>
<223>Multiple PCR primer（EGFR-20-No.2-R）
<400> 12
attgtctttg tgttcccgga c 21
<210> 13
<211> 17
<212> DNA
<213> Artificial
<220>
<223>Multiple PCR primer（EGFR-20-No.3-F）
<400> 13
gcagctcatg cccttcg 17
<210> 14
<211> 21
<212> DNA
<213> Artificial
<220>
<223>Multiple PCR primer（EGFR-20-No.3-R）
<400> 14
cgtatctccc ttccctgatt a 21
<210> 15
<211> 20
<212> DNA
<213> Artificial
<220>
<223>Multiple PCR primer（EGFR-21-No.1-F）
<400> 15
tgaactactt ggaggaccgt 20
<210> 16
<211> 20
<212> DNA
<213> Artificial
<220>
<223>Multiple PCR primer（EGFR-21-No.1-R）
<400> 16
ccttactttg cctccttctg 20
<210> 17
<211> 20
<212> DNA
<213> Artificial
<220>
<223>Multiple PCR primer（EGFR-21-No.2-F）
<400> 17
aaacaccgca gcatgtcaag 20
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<211> 21
<212> DNA
<213> Artificial
<220>
<223>Multiple PCR primer（EGFR-21-No.2-R）
<400> 18
aaatgctggc tgacctaaag c 21
<210> 19
<211> 21
<212> DNA
<213> Artificial
<220>
<223>Multiple PCR primer（EGFR-21-No.4-F）
<400> 19
attcggatgc agagcttctt c 21
<210> 20
<211> 19
<212> DNA
<213> Artificial
<220>
<223>Multiple PCR primer（EGFR-21-No.4-R）
<400> 20
gatcttgaca tgctgcggt 19
<210> 21
<211> 20
<212> DNA
<213> Artificial
<220>
<223>Multiple PCR primer（KRAS-2-No.1-F）
<400> 21
cgtcaaggca ctcttgccta 20
<210> 22
<211> 21
<212> DNA
<213> Artificial
<220>
<223>Multiple PCR primer（KRAS-2-No.1-R）
<400> 22
ggtactggtg gagtatttga t 21
<210> 23
<211> 20
<212> DNA
<213> Artificial
<220>
<223>Multiple PCR primer（KRAS-2-No.4-F）
<400> 23
ggtcctgcac cagtaatatg 20
<210> 24
<211> 25
<212> DNA
<213> Artificial
<220>
<223>Multiple PCR primer（KRAS-2-No.4-R）
<400> 24
aatataaact tgtggtagtt ggagc 25
<210> 25
<211> 22
<212> DNA
<213> Artificial
<220>
<223>Multiple PCR primer（BRAF-15-No.1-F）
<400> 25
gactttctag taactcagca gc 22
<210> 26
<211> 21
<212> DNA
<213> Artificial
<220>
<223>Multiple PCR primer（BRAF-15-No.1-R）
<400> 26
cagtgaaatc tcgatggagt g 21
<210> 27
<211> 23
<212> DNA
<213> Artificial
<220>
<223>Multiple PCR primer（BRAF-15-No.2-F）
<400> 27
ccagacaact gttcaaactg atg 23
<210> 28
<211> 22
<212> DNA
<213> Artificial
<220>
<223>Multiple PCR primer（BRAF-15-No.2-R）
<400> 28
gctctgatag gaaaatgaga tc 22
<210> 29
<211> 65
<212> DNA
<213> Artificial
<220>
<223>Adaptor sequences
<220>
<221> misc_feature
<222> (1)..(1)
<223>Phosphorylation modification
<220>
<221> misc_feature
<222> (32)..(32)
<223>Deoxidation is modified
<220>
<221> misc_feature
<222> (65)..(65)
<223>Phosphorothioate
<400> 1
gatcggaaga gcacacgtct gaactccagt cuacactctt tccctacacg acgctcttcc 60
gatct
Claims (10)
1. a kind of multiple PCR primer, it is characterised in that the multiple PCR primer is made up of following primer pair：
SEQ ID NO:1 and SEQ ID NO:Primer pair shown in 2, SEQ ID NO:5 and SEQ ID NO:Primer pair shown in 6, SEQ
ID NO:9 and SEQ ID NO:Primer pair shown in 10, SEQ ID NO:13 and SEQ ID NO:Primer pair shown in 14, SEQ ID
NO:15 and SEQ ID NO:Primer pair shown in 16 and SEQ ID NO:19 and SEQ ID NO:Primer pair shown in 20.
2. the multiple PCR primer described in claim 1, it is characterised in that the multiple PCR primer is also included in following primer pair
At least one pair of：SEQ ID NO:3 and SEQ ID NO:Primer pair shown in 4, SEQ ID NO:7 and SEQ ID NO:Draw shown in 8
Thing pair, SEQ ID NO:11 and SEQ ID NO:Primer pair shown in 12, SEQ ID NO:17 and SEQ ID NO:Primer shown in 18
It is right, SEQ ID NO:21 and SEQ ID NO:Primer pair shown in 22, SEQ ID NO:23 and SEQ ID NO:Primer pair shown in 24,
SEQ ID NO:25 and SEQ ID NO:Primer pair shown in 26 and SEQ ID NO:27 and SEQ ID NO:Primer pair shown in 28.
3. the multiple PCR primer described in claim 1 or 2 obtain and/or detect EGFR gene sequence and detection EGFR,
Purposes in KRAS and/or BRAF gene sequence.
4. a kind of test kit, it includes the multiple PCR primer described in claim 1 or 2.
5. the test kit described in claim 4 is obtaining and/or is detecting EGFR gene sequence and expanding and/or detecting
Purposes in EGFR, KRAS and/or BRAF gene sequence.
6. a kind of method of amplification gene sequence target area, it is characterised in that methods described is included using claim 1 or 2
Described multiple PCR primer carries out the amplification.
7. the method that a kind of EGFR, KRAS and/or BRAF gene are detected, it is characterised in that include：
(1) at least a portion nucleic acid in testing sample is expanded using the method described in claim 6, obtains amplification and produce
Thing；
(2) amplified production is analyzed, to obtain EGFR, KRAS and/or BRAF gene testing result.
8. the method described in claim 7, it is characterised in that the nucleic acid is DNA and/or RNA.
9. the method described in claim 7, it is characterised in that the nucleic acid is RNA, step (1) includes：
(1-1) it is cDNA by the RNA reverse transcriptions using the upstream in the multiple PCR primer or downstream primer；And
(1-2) cDNA is expanded using corresponding downstream in the multiple PCR primer or forward primer, is expanded
Product.
10. the method described in claim 7, it is characterised in that step (2) includes：
(2-1) amplified production is sequenced；And
(2-2) by sequencing result respectively with EFGR, KRAS and/or BRAF gene wild-type sequence is compared, to determine EFGR, KRAS
And/or the mutational site of BRAF gene.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710146087.7A CN106676105A (en) | 2015-11-04 | 2015-11-04 | Primers capable of simultaneously acquiring and detecting multiple target area sequences, kit and method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710146087.7A CN106676105A (en) | 2015-11-04 | 2015-11-04 | Primers capable of simultaneously acquiring and detecting multiple target area sequences, kit and method |
| CN201510740906.1A CN105331611A (en) | 2015-11-04 | 2015-11-04 | Multi-PCR primer for detecting EGFR/KRAS/BRAF genetic mutation locus based on next-generation sequencing technology and method and application |
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| CN201510740906.1A Division CN105331611A (en) | 2015-11-04 | 2015-11-04 | Multi-PCR primer for detecting EGFR/KRAS/BRAF genetic mutation locus based on next-generation sequencing technology and method and application |
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| CN201710121139.5A Pending CN106868139A (en) | 2015-11-04 | 2015-11-04 | PCR primer, kit and purposes |
| CN201710132480.0A Pending CN106801104A (en) | 2015-11-04 | 2015-11-04 | Multiple PCR primer, kit and purposes |
| CN201710146087.7A Pending CN106676105A (en) | 2015-11-04 | 2015-11-04 | Primers capable of simultaneously acquiring and detecting multiple target area sequences, kit and method |
| CN201510740906.1A Pending CN105331611A (en) | 2015-11-04 | 2015-11-04 | Multi-PCR primer for detecting EGFR/KRAS/BRAF genetic mutation locus based on next-generation sequencing technology and method and application |
| CN201710145470.0A Pending CN106834283A (en) | 2015-11-04 | 2015-11-04 | Primer, kit and the method for multiple target areas sequences are obtained and detected simultaneously |
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| CN201710121139.5A Pending CN106868139A (en) | 2015-11-04 | 2015-11-04 | PCR primer, kit and purposes |
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| CN201710145470.0A Pending CN106834283A (en) | 2015-11-04 | 2015-11-04 | Primer, kit and the method for multiple target areas sequences are obtained and detected simultaneously |
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| CN (5) | CN106868139A (en) |
| HK (1) | HK1220723A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106868139A (en) * | 2015-11-04 | 2017-06-20 | 深圳市瀚海基因生物科技有限公司 | PCR primer, kit and purposes |
| CN105886650A (en) * | 2016-06-08 | 2016-08-24 | 广州凯普医药科技有限公司 | KRAS gene mutation detection kit |
| CN107630075A (en) * | 2016-07-18 | 2018-01-26 | 丰技生物科技股份有限公司 | The detection method in the mutational site of EGFR gene |
| CN107488729A (en) * | 2017-09-27 | 2017-12-19 | 辽宁科骏生物有限公司 | Detect Primer composition, kit and the method for EGFR genetic mutation |
| CN107988369B (en) * | 2017-12-28 | 2020-11-17 | 北京雅康博生物科技有限公司 | Kit for simultaneously detecting 45 mutation sites of human EGFR gene |
| CN111518901A (en) * | 2020-04-30 | 2020-08-11 | 北京和合医学诊断技术股份有限公司 | Primer group for detecting EGFR gene mutation and application method thereof |
| CN111500727A (en) * | 2020-04-30 | 2020-08-07 | 北京和合医学诊断技术股份有限公司 | Primer group for detecting KRAS gene and BRAF gene mutation and application method thereof |
| CN112899366A (en) * | 2021-01-06 | 2021-06-04 | 南京普济生物有限公司 | Method and kit for detecting colorectal cancer gene mutation site |
| CN117106911A (en) * | 2023-08-14 | 2023-11-24 | 首都医科大学附属北京天坛医院 | Gene set for detecting schwannoma and multiplex PCR-high flux sequencing detection kit thereof |
| CN117844933B (en) * | 2024-03-07 | 2024-06-11 | 上海复迪生生命科学有限公司 | Multiplex PCR primer group for detecting lung tumor related gene variation and application thereof |
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| Publication number | Publication date |
|---|---|
| HK1220723A1 (en) | 2017-05-12 |
| CN106834283A (en) | 2017-06-13 |
| CN106868139A (en) | 2017-06-20 |
| CN106801104A (en) | 2017-06-06 |
| WO2017076300A1 (en) | 2017-05-11 |
| CN105331611A (en) | 2016-02-17 |
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