CN107304442A - Detect primer pair, kit and the method for the exon codon mutation of KRAS genes 4 - Google Patents
Detect primer pair, kit and the method for the exon codon mutation of KRAS genes 4 Download PDFInfo
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Abstract
The invention discloses primer pair, kit and the method for a kind of detection KRAS gene codons mutation, the primer pair is the SEQ ID NO for No. 117 codon mutation detections of 4 exon:1 and SEQ ID NO:The 2, and/or SEQ ID NO for No. 146 codon mutation detections of 4 exon:3 and SEQ ID NO:4, detection method is, using the DNA of detected sample as template, to carry out quantitative fluorescent PCR reaction, collects fluorescence signal, draws melting curve；The melting curve of interpretation detected sample.The detection method of the present invention inexpensively, quickly, can be detected sensitively to the exon 117 of KRAS genes 4 and No. 146 codon mutations, be adapted to clinical molecular diagnosis laboratory and used.
Description
Technical field
The invention belongs to biomedicine technical field, specifically, KRAS genes are detected the present invention relates to one kind
Primer pair, kit and the method for 4 No. 117 and No. 146 codon mutations of exon.
Background technology
KRAS genes are a kind of proto-oncogenes, are about 35kb, positioned at No. 12 chromosomes, are ras genes men
One of race member, encoded K RAS albumen.It is one important Downstream regulatory of EGFR signal transduction pathways
Gene, KRAS genes receive signal when undergoing mutation without EGFR, the auto-activation path and can start
The signal transduction in downstream, causes Cellular Signaling Transduction Mediated disorderly, uncontrolled cellular proliferation.
With the fast development of modern medicine, the individualized treatment of molecular targeted agents is used widely.2008
Year, National Cancer complex treatment alliance (NCCN) exists《Colorectal cancer clinical practice guideline》In first
Propose that EGFR targeting medicament curative effects and KRAS gene appearances are closely related.2011, NCCN existed《Knot
Carcinoma of the rectum clinical practice guideline》(V3.2011) explicitly point out：(1) all metastatic colorectal cancer patients should all be examined
Survey KRAS gene appearances；(2) KRAS gene wild type patients just advise receiving EGFR inhibitor (such as
Cetuximab and Victibix) treatment., NCCN pairs in 2015《Colorectal cancer clinical practice guideline》
(V2.2015) update：Detect KRAS gene appearances, including KRAS exon 2s and exon 3,4
And the exon 2 of NRAS genes, 3 and 4, in addition, also needing to detect BRAF gene state, either
It is no to have KRAS.
4 exon No. 117 and No. 146 of KRAS genes is as hot mutant site, more and more clinics
Laboratory starts to carry out the item detection.Method more common at present includes：PCR sequencing PCR, based on the glimmering of probe
Fluorescent Quantitative PCR method etc..PCR sequencing PCR is presently believed to be because the change of each bases of DNA can be read
The goldstandard method of gene mutation is detected, but because sensitivity is low, sequencing steps are cumbersome, time-consuming, pair set
The requirement of standby and operating personnel is higher, is not easy to be formed the molecule diagnostic products of normalizing operation.Based on probe
Fluorescence quantitative PCR method detection gene mutation realize that this method is sensitive by known specific probe
Degree is high, specificity is good, at present the existing commercial kit of the detection KRAS gene mutation based on this method,
But this method is directed to the rare mutation occurred on No. 117 and No. 146 codons of 4 exon of KRAS genes
Type, then can not be detected, and detection reagent cost is high.
Therefore, it is necessary to a kind of improved method is provided with realize KRAS genes 4 exon 117 and
Quick, the accurate and cheap detection method of No. 146 codon mutation states.
The content of the invention
Based on this, in order to overcome the defect of above-mentioned prior art, the invention provides one kind for detection KRAS
Primer pair, kit and the HRM detection sides of 4 No. 117 and No. 146 codon mutations of exon of gene
Method.
In order to realize foregoing invention purpose, this invention takes following technical scheme：
A kind of primer pair of the exon codon mutation of detection KRAS genes 4, the primer pair is：For
The SEQ ID NO of No. 117 codon mutation detections:1 and SEQ ID NO:2, and/or for No. 146 passwords
The SEQ ID NO of sub- abrupt climatic change:3 and SEQ ID NO:4.
It is described present invention also offers a kind of kit of the exon codon mutation of detection KRAS genes 4
Kit is included for the detection of No. 117 codon mutations such as SEQ ID NO:1 and SEQ ID NO:Shown in 2
Primer pair, and/or for No. 146 codon mutations detection such as SEQ ID NO:3 and SEQ ID NO:4 institutes
The primer pair shown.
In wherein some embodiments, the detection kit also includes saturated fluorescence dyestuff.
In wherein some embodiments, the saturated fluorescence dyestuff is Eva Green.
In wherein some embodiments, the detection kit also includes Buffer, dNTP and Taq enzyme.
Present invention also offers a kind of reaction system of the exon codon mutation of detection KRAS genes 4, institute
Stating reaction system is included for the detection of No. 117 codon mutations such as SEQ ID NO:1 and SEQ ID NO:2
Shown primer pair, and/or for No. 146 codon mutations detections such as SEQ ID NO:3 and SEQ ID
NO:Primer pair shown in 4.
In wherein some embodiments, the reaction system also includes Eva Green, dNTP and Taq enzyme.
In wherein some embodiments, the reaction system for No. 117 codon mutation detections is：DNA moulds
Plate 1-5 μ L, 10 × Buffer 2-2.5 μ L, dNTP 2-2.5 μ L, Eva Green 1-1.25 μ L, Mg2+0.8-1.5μL、
SEQ ID NO:1 primer 0.6-1 μ L, SEQ ID NO:2 primer 0.6-1 μ L, Taq enzyme 0.25-0.4 μ L, nothing
Enzyme water adds to 20-25 μ L；Reaction system for the detection of No. 146 codon mutations is：DNA profiling 1-5 μ L,
10×Buffer 2-2.5μL、dNTP 2-2.5μL、Eva Green 1-1.25μL、Mg2+0.8-1.5μL、SEQ ID
NO:3 primer 0.6-1 μ L, SEQ ID NO:4 primer 0.6-1 μ L, Taq enzyme 0.25-0.4 μ L, add to without enzyme water
20-25μL。
Present invention also offers a kind of method of the exon codon mutation of detection KRAS genes 4, including with
Lower step：
(1), using the DNA of detected sample as template, using for the detection of No. 117 codon mutations as
SEQ ID NO:1 and SEQ ID NO:Primer pair shown in 2, and/or detected for No. 146 codon mutations
Such as SEQ ID NO:3 and SEQ ID NO:Primer pair shown in 4 carry out respectively quantitative fluorescent PCR reaction and
HRM is analyzed, and collects fluorescence signal；
(2), the melting curve of interpretation detected sample, if detected sample for No. 117 codon mutations
Melting curve in show the melting peakss of two and the above, then it represents that No. 117 codons of the patient are present
Mutation, such as only shows a melting peakss, then it represents that No. 117 codons of the patient are wild type.Similarly,
If showing the melting of two and the above in the melting curve for No. 146 codon mutations of detected sample
Peak, then it represents that No. 146 codons of the patient have mutation, such as only show a melting peakss, then it represents that should
No. 146 codons of patient are wild type.
In wherein some embodiments, quantitative fluorescent PCR of the step (1) for No. 117 codon mutation detections
Reaction system be：DNA profiling 1-5 μ L, 10 × Buffer 2-2.5 μ L, dNTP 2-2.5 μ L, Eva Green
1-1.25μL、Mg2+0.8-1.5μL、SEQ ID NO:1 primer 0.6-1 μ L, SEQ ID NO:2 primer 0.6-1 μ L,
Taq enzyme 0.25-0.4 μ L, 20-25 μ L are added to without enzyme water；Determine for the fluorescence of No. 146 codon mutation detections
Amount PCR reaction system be：DNA profiling 1-5 μ L, 10 × Buffer 2-2.5 μ L, dNTP 2-2.5 μ L,
Eva Green 1-1.25μL、Mg2+0.8-1.5μL、SEQ ID NO:3 primer 0.6-1 μ L, SEQ ID NO:4
Primer 0.6-1 μ L, Taq enzyme 0.25-0.4 μ L, 20-25 μ L are added to without enzyme water.
In wherein some embodiments, the response procedures of step (1) described quantitative fluorescent PCR are：95℃5
Min → 95 DEG C 10s, 62 DEG C of 15s, 72 DEG C of 25s, 40cycles → 95 DEG C 1min → 40 DEG C 1min → melting temperature
72-83 DEG C of degree, temperature often raises 1 DEG C, obtains 15 fluorescence signal → 40 DEG C 10s.
In wherein some embodiments, the concentration of step (1) described template is 50-100ng/ μ L.
Compared with prior art, the invention has the advantages that：
1st, the present inventor passes through multiple exploration discovery, and KRAS bases are carried out using the kit of the present invention
Because of 4 exons 117 and No. 146 codon HRM methods detections, as little as 5% mutating molecule is can detect that,
All mutation types on the exon 117 of KRAS genes 4 and No. 146 codons can be detected, not by prominent
Become base position with type to limit to；Compared with Sanger PCR sequencing PCRs (goldstandard method) result, as a result unanimously
Rate is 99.84% (657/658)；Unique 1 sample not being inconsistent is dashed forward for 19 kinds through Xiamen Ai De KRAS genes
Become detection kit detection, its result is consistent with the result of the inventive method.Illustrate that this sample is actually
Belong to low abundance mutation, goldstandard Sanger PCR sequencing PCRs can not be detected because detection sensitivity is low, and of the invention
Sensitivity it is high, the sample being mutated for low abundance can also be detected.Separately there is the DNA of 1 sample of poor quality,
PCR sequencing PCR can not obtain testing result, but utilize the inventive method, can obtain testing result, and and Xiamen
19 kinds of mutation detection kit testing results of Ai De KRAS genes are consistent.Illustrate the inventive method to sample
DNA quality requirement is low, and applicability is wide.Meanwhile, requirement of the inventive method to equipment is greatly reduced,
Without using sequenator, a quantitative real time PCR Instrument for carrying HRM functions, the scope of application are only needed
Wider, instrument cost is lower；
2nd, detection method operation sequence of the invention is greatly simplified, whole stopped pipe operation, it is to avoid cross pollution,
The external diagnosis reagent product of normalizing operation is easily formed, and detection time and reagent cost are substantially reduced, 60-90
Detection can be completed in minute.
Brief description of the drawings
Fig. 1 be sample in contain No. 117 codon AAA of 4 exon of KRAS genes>AAT mutation
HRM analysis charts；
Fig. 2 be sample in be free of No. 117 codon AAA of 4 exon of KRAS genes>AAT mutation
HRM analysis charts；
Fig. 3 be sample in contain No. 146 codon GCA of 4 exon of KRAS genes>CCA mutation
HRM analysis charts；
Fig. 4 be sample in be free of No. 146 codon GCA of 4 exon of KRAS genes>CCA mutation
HRM analysis charts；
Fig. 5 is No. 117 K117N (AAA of 4 exon of KRAS genes of different mutant proportions>AAC) dash forward
Become the HRM analysis charts of standard items 1；Wherein, A is 25% mutation standard items 1, and B is 10% mutation standard
Product 1, C is 5% mutation standard items 1, and D is 0% mutation standard items 1；
Fig. 6 is No. 146 A146V (GCA of 4 exon of KRAS genes of different mutant proportions>GTA)
It is mutated the HRM analysis charts of standard items 2；Wherein, A is 25% mutation standard items 2, and B is 10% mutation mark
Quasi- product 2, C is 5% mutation standard items 2, and D is 0% mutation standard items 2；
Fig. 7 detects KRAS genes 4 to use different primers using HRM detection methods of the present invention
The amplification curve of No. 17 codon mutations of exons 1, wherein, it is good using the expanding effect of primer pair 1, can
Correctly to tell mutant sample；Saltant type sample can not be detected using primer pair 3,4；Use primer pair
5th, 6 effects are poor；
Fig. 8 detects KRAS genes 4 to use different primers using HRM detection methods of the present invention
The amplification curve of No. 46 codon mutations of exons 1, wherein, it is good using the expanding effect of primer pair 2, can
Correctly to tell mutant sample；Saltant type sample can not be detected using primer pair 7,8；Use primer pair
9th, 10 effects are poor.
Embodiment
Technical scheme is further illustrated below by way of specific embodiment, specific embodiment is not represented
Limiting the scope of the invention.Some nonessential modifications that other people are made according to theory of the present invention
Protection scope of the present invention is still fallen within adjustment.
Step in following examples is this area Conventional procedures in addition to specified otherwise, real below
The raw material used in example is applied, is derived from commercially available.
The mutation of the intestinal cancer neoplasmic tissue sample KRAS of embodiment 14 No. 117 and No. 146 codons of exon
Detection
1st, primer
A kind of detection intestinal cancer neoplasmic tissue sample KRAS of the present invention No. 117 codons of 4 exon
AAA>The primer of AAT mutation, the base sequence with SEQ ID NO.1 and SEQ ID NO.2.
Sense primer SEQ ID NO.1:AGGACTCTGAAGATGTACCTAT
Anti-sense primer SEQ ID NO.2:AGGACTCTGAAGATGTACCTAT
A kind of No. 146 codon GCA of 4 exon for detecting intestinal cancer neoplasmic tissue sample KRAS>CCA
Mutation primer, the base sequence with SEQ ID NO.3 and SEQ ID NO.4.
Sense primer SEQ ID NO.3:CACAAAACAGGCTCAGGA
Anti-sense primer SEQ ID NO.4:CAGTGTTACTTACCTGTCTTGTC
2nd, reaction system
Reactant is formulated as follows respectively using Blend Taq Plus enzymes (Toyobo, CAT NO.BTQ-201)
System：
| Title | Consumption (μ L) |
| 10×Buffer | 2 |
| dNTP | 2 |
| EvaGreen | 1 |
| Mg2+ | 0.8 |
| SEQ ID NO:1 primer | 0.6 |
| SEQ ID NO:2 primers | 0.6 |
| Taq enzyme | 0.25 |
| Without enzyme water | 11.75 |
| DNA profiling | 1 |
3rd, detection method
(1), sample source and extracting genome DNA：All patients with bowel cancer tumor samples are all from Zhongshan University
Attached 6th hospital pathology department, collects tissue, using U.S. base paraffinized sample extracts kit to genomic DNA
Extracted.DNA profiling concentration is separately adjusted to angularly 50-100ng/ul.In above-mentioned two reaction system respectively
Add 1ul DNA profiling.It is vortexed after mixing, is determined respectively using the real-time fluorescences of Roche LightCycler 480
Amount PCR instrument is detected that program is as follows：95℃5min→(95℃10s→62℃15s→72℃25s)
40cycles (being arranged on 72 DEG C of collection fluorescence signals) → 95 DEG C of 1min → 40 DEG C 1min → melting temperatures
(72-83 DEG C), temperature often raises 1 DEG C, obtains 15 fluorescence signal → 40 DEG C 10s.
(2), the melting curve of interpretation sample to be measured, with determine No. 117 of the exon of KRAS genes 4 and
No. 146 codons are with the presence or absence of mutation.If No. 117 codons of the exon of sample KRAS genes 4 are present
AAA>AAT is mutated, then testing sample melting curve shows the melting peakss of two and the above, and 100 are detected altogether
Example sample, results contrast is carried out with Sanger PCR sequencing PCRs, and two methods result 100% is consistent.If sample KRAS
There is GCA in No. 146 codons of the exon of gene 4>CCA is mutated, then testing sample melting curve shows
The melting peakss of two and the above are shown, 100 samples are detected altogether, results contrast is carried out with Sanger PCR sequencing PCRs,
Two methods result 100% is consistent.
Fig. 1 be sample in contain No. 117 codon AAA of 4 exon of KRAS genes>AAT mutation
HRM analysis charts；Fig. 2 be sample in be free of No. 117 codon AAA of 4 exon of KRAS genes>AAT
The HRM analysis charts of mutation.Fig. 3 be sample in contain KRAS genes No. 146 codons of 4 exon
GCA>The HRM analysis charts of CCA mutation；Fig. 4 be sample in be free of the exon 146 of KRAS genes 4
Number codon GCA>The HRM analysis charts of CCA mutation.
The kit of the mutation of the detection of embodiment 2 KRAS 4 No. 117 and No. 146 codons of exon
The detection kit of the present embodiment includes following reagent 1 and reagent 2, respectively including following component：
| Title | Consumption (μ L) |
| 10×Buffer | 2 |
| dNTP | 2 |
| EvaGreen | 1 |
| Mg2+ | 0.8 |
| SEQ ID NO:1 primer | 0.6 |
| SEQ ID NO:2 primers | 0.6 |
| Taq enzyme | 0.25 |
| Without enzyme water | 11.75 |
| DNA profiling | 1 |
| Title | Consumption (μ L) |
| 10×Buffer | 2 |
| dNTP | 2 |
| EvaGreen | 1 |
| Mg2+ | 0.8 |
| SEQ ID NO:3 primers | 0.6 |
| SEQ ID NO:4 primers | 0.6 |
| Taq enzyme | 0.25 |
| Without enzyme water | 11.75 |
| DNA profiling | 1 |
The sensitivity checking of the inventive method of test example 1
Sensitivity checking takes following methods：
1st, using No. 117 K117N (AAA of 4 exon of KRAS genes>AAC) it is mutated the He of standard items 1
No. 146 A146V (GCA>GTA standard items 2) are mutated, are configured to respectively with wild type standard items following several
The standard items 1 and 2 of different mutant proportions：25% mutation, 10% mutation, 5% mutation, 0% mutation, specifically
Compound method refers to the conventional method of the art.
2nd, two reaction systems in detection reagent 1 and 2, i.e. embodiment 1 are prepared；
3rd, standard items 1, the 2 each 1ul for the different mutant proportions that step 1 prepared are taken, step 2 is separately added into
Middle detection reagent 1 and detection reagent 2, the real time fluorescent quantitatives of Roche LightCycler 480 are put into by detection reagent
Detected in PCR instrument；
4th, PCR and HRM programs：95℃5min→(95℃10s→62℃15s→72℃25s)
40cycles (being arranged on 72 DEG C of collection fluorescence signals) → 95 DEG C of 1min → 40 DEG C 1min → melting temperatures
(72-83 DEG C), temperature often raises 1 DEG C, obtains 15 fluorescence signal → 40 DEG C 10s.
5th, HRM interpretations of result：If there is mutation in No. 117 codons of the exon of sample KRAS genes 4,
Then the testing sample melting curve of detection reagent 1 shows the melting peakss of two and the above；If sample KRAS
There is mutation in No. 146 codons of the exon of gene 4, then the testing sample melting curve of detection reagent 2
Show the melting peakss of two and the above.
As a result as shown in Figure 5 and Figure 6.Fig. 5 is the exon 117 of KRAS genes 4 of different mutant proportions
Number K117N (AAA>AAC the HRM analysis charts of standard items 1) are mutated, as seen from Figure 5, this hair are used
Bright detection reagent 1 and detection method guarantee the mutation of detection 5%.Fig. 6 is the KRAS of different mutant proportions
No. 146 A146V (GCA of 4 exon of gene>GTA the HRM analysis charts of standard items 2) are mutated, by scheming
6 be can be seen that, the mutation of detection 5% is guaranteed using the detection reagent 2 and detection method of the present invention.
19 kinds of abrupt climatic changes of the inventive method of test example 2 and Sanger PCR sequencing PCRs and Xiamen Ai De KRAS genes are tried
The testing result of agent box compares
659 clinical samples are all from ZhongShan University attached No.6 Hospital pathology department, wherein colon cancer 320,
The carcinoma of the rectum 277, stomach cancer 55, carcinoma of anal canal 7.The method of the embodiment of the present invention 1 or 2, Sanger are taken respectively
PCR sequencing PCR and Xiamen 19 kinds of mutation detection kits of Ai De KRAS genes are detected to this 659 samples,
As a result it is as shown in table 1.
The testing result of 1 three kinds of methods of table compares
As seen from the results in Table 1,658 samples are had while having the method for the invention and goldstandard Sanger
The result of PCR sequencing PCR, the specificity of the inventive method is 99.84%, and sensitiveness is 100%, and positive predictive value is
94.44%.The sample that unique 1 result is not consistent, is tried through 19 kinds of abrupt climatic changes of Xiamen Ai De KRAS genes
After agent box is detected, its result is consistent with the method for the invention, and it is actually category to illustrate this sample
In the mutation of low abundance, goldstandard Sanger PCR sequencing PCRs can not be detected because detection sensitivity is low, and the present invention
Sensitivity is high, and the sample being mutated for low abundance can also be detected.
Separately there is 1 sample because DNA is of poor quality, can not be detected with Sanger PCR sequencing PCRs, and present invention side
Method can detect that testing result is wild type to it.Through the 19 kinds of mutation inspections of Xiamen Ai De KRAS genes
After test agent box is detected, its result is consistent with the method for the invention, illustrates the inventive method to sample
This DNA quality requirement is low, and applicability is wide.
Test example 3 is using Comparative result of the different primers to the abrupt climatic change of the exon of KRAS genes 4
Inventor has groped multigroup primer pair, and research HRM methods (methods of Examples 1 and 2) are above-mentioned to detecting
The influence of the experimental result of the exon gene mutation of KRAS genes 4.Table 2 below is KRAS 4 extras
It is real that No. 117 codons of aobvious son use multigroup Exemplary primers to be carried out using detection reagent 1 in the method for embodiment 1
Test acquired results.It is demonstrated experimentally that the final choice of primer concerns the feasibility of method.
Table 2 is compared using 5 pairs of primer pairs and peak type, sentence read result
Note：Upper table uses HRM detection methods, and its PCR program sets 40 circulations altogether.Sample amplification CT values<25 are preferred,
If sample amplification CT values are not within the range, interpretation is that this PCR reaction expanding effect is bad or can not expand, and is finally had
The amount of amplified production and follow-up HRM analyses may be influenceed.
As a result show, sample 1 is saltant type using the interpretation of Sanger PCR sequencing PCRs, and 5 pairs of primers in table 2
Detected using the method for the invention and detection reagent 1.It was found that only SEQ ID NO:1、SEQ ID NO:2
It is saltant type, remaining 4 pairs that shown primer (primer pair 1 in i.e. upper list 2), which can accurately detect sample 1,
Primer (primer pair 3-6 in i.e. upper list) can not accurately interpretation.
Table 3 below uses detection for KRAS No. 146 codons of 4 exon using multigroup Exemplary primers
Reagent 2 carries out experiment acquired results in the method for embodiment 1.It is demonstrated experimentally that the final choice side of concerning of primer
The feasibility of method.
Table 3 is compared using 5 pairs of primer pairs and peak type, sentence read result
Note：Upper table uses HRM detection methods, and its PCR program sets 40 circulations altogether.Sample amplification CT values<25 are preferred,
If sample amplification CT values are not within the range, interpretation is that this PCR reaction expanding effect is bad or can not expand, and is finally had
The amount of amplified production and follow-up HRM analyses may be influenceed.
As a result show, sample 2 is saltant type using the interpretation of Sanger PCR sequencing PCRs, and 5 pairs of primers in table 3
Detected using HRM methods of the present invention and detection reagent 1.It was found that only SEQ ID NO:1、SEQ ID
NO:It is saltant type that primer shown in 2 (primer pair 2 in i.e. upper list 4), which can accurately detect sample 2, its
Remaininging 4 pairs of primers (primer pair 7-10 in i.e. upper list) can not accurately interpretation.
The detection method of test example 4 and Sanger PCR sequencing PCR times and Cost comparisons
Computational methods：For thering is the 657 of testing result to treat mark sheet using two methods in test example 2
Adjusted, as a result as shown in table 4.
The comparison of the inventive method of table 4. and the detection time and cost of Sanger PCR sequencing PCR kits
| HRM methods of the present invention | Sanger PCR sequencing PCRs | |
| Experiment spends time estimation | 1.5 hours/sample | 10.5 hours/sample |
| Experimental cost is estimated | 7 yuan/sample | 35 yuan/sample |
From result, 4 exons 117 and 146 of the HRM detection methods of the present invention to KRAS are used
Site is detected that the detection time of each sample shortens 85.71% than Sanger PCR sequencing PCR, while each
The testing cost of sample reduces 80%.4 extras using HRM methods of the present invention for KRAS show
The detection in sub 117 and 146 sites, can greatly save detection time and cost, more preferable clinical service disease
People.
Embodiment described above only expresses the several embodiments of the present invention, and it describes more specific and detailed,
But can not therefore it be construed as limiting the scope of the patent.It should be pointed out that for this area
For those of ordinary skill, without departing from the inventive concept of the premise, some deformations can also be made and changed
Enter, these belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be with appended power
Profit requires to be defined.
Claims (8)
1. a kind of primer pair of the exon codon mutation of detection KRAS genes 4, it is characterised in that institute
Stating primer pair is：For the SEQ ID NO of No. 117 codon mutation detections:1 and SEQ ID NO:2, and/
Or for the SEQ ID NO of No. 146 codon mutation detections:3 and SEQ ID NO:4.
2. a kind of kit of the exon codon mutation of detection KRAS genes 4, it is characterised in that institute
Stating kit includes the primer pair described in claim 1.
3. the kit of the exon codon mutation of detection KRAS genes 4 according to claim 2,
Characterized in that, the kit also includes saturated fluorescence dyestuff.
4. the kit of the exon codon mutation of detection KRAS genes 4 according to claim 3,
Characterized in that, the saturated fluorescence dyestuff is Eva Green.
5. a kind of method of the exon codon mutation of detection KRAS genes 4, it is characterised in that including
Following steps：
(1), using the DNA of detected sample as template, entered respectively using the primer pair shown in claim 1
Row quantitative fluorescent PCR reacts, and collects fluorescence signal, draws melting curve；
(2), the melting curve of interpretation detected sample.
6. the method for the exon codon mutation of detection KRAS genes 4 according to claim 5,
Characterized in that, step (1) is for the reaction system of the quantitative fluorescent PCR of No. 117 codon mutation detections：
DNA profiling 1-5 μ L, 10 × Buffer 2-2.5 μ L, dNTP 2-2.5 μ L, Eva Green 1-1.25 μ L, Mg2+
0.8-1.5μL、SEQ ID NO:1 primer 0.6-1 μ L, SEQ ID NO:2 primer 0.6-1 μ L, Taq enzyme
0.25-0.4 μ L, 20-25 μ L are added to without enzyme water；For the quantitative fluorescent PCR of No. 146 codon mutation detections
Reaction system be：DNA profiling 1-5 μ L, 10 × Buffer 2-2.5 μ L, dNTP 2-2.5 μ L, Eva Green
1-1.25μL、Mg2+0.8-1.5μL、SEQ ID NO:3 primer 0.6-1 μ L, SEQ ID NO:4 primer 0.6-1 μ L,
Taq enzyme 0.25-0.4 μ L, 20-25 μ L are added to without enzyme water.
7. the method for the exon codon mutation of detection KRAS genes 4 according to claim 5,
Characterized in that, the response procedures of step (1) described quantitative fluorescent PCR are：95℃5min→95℃10s、
62 DEG C of 15s, 72 DEG C of 25s, 40cycles → 95 DEG C 1min → 40 DEG C 1min → 72-83 DEG C of melting temperature, temperature
Degree often raises 1 DEG C, obtains 15 fluorescence signal → 40 DEG C 10s.
8. the method for the exon codon mutation of detection KRAS genes 4 according to claim 5,
Characterized in that, the concentration of step (1) described template is 50-100ng/ μ L.
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