CN106048023B - Diagnostic Kit and method for detecting mutation of exon 13 of human C-Kit gene - Google Patents
Diagnostic Kit and method for detecting mutation of exon 13 of human C-Kit gene Download PDFInfo
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Abstract
The invention provides a kit for detecting human c-kit gene mutation types, which comprises a primer pair shown in SEQ ID NO. 1-4 and a probe shown in SEQ ID NO. 5. The invention also discloses a method for detecting the mutation of the exon 13 of the human c-kit gene. The kit and the method can accurately detect the K642E, V654A and N655K mutation of the exon 13 of the human c-kit gene, provide guidance for the medication of GIST patients and have good clinical application prospect.
Description
Technical field
The invention belongs to genetic test fields, and in particular to a kind of to detect what 13 exon of mankind C-Kit gene was mutated
Diagnostic kit and method.
Background technique
Gastrointestinal stromal tumor (gastrointestinal stromaltumors, GIST) is a kind of originating between gastrointestinal tract
The tumour of leaf texture is most common leaf source property tumour of gastrointestinal tract, is mainly in middle-older patient, men and women's disease incidence is without obvious poor
It is different.Mazur etc. proposes that GIST is a kind of independent tumors type for being different from liomyoma and neurogenic tumour in nineteen eighty-three,
Its direct cause of disease is the acquired function mutation of oncogene.
Early treatment GIST relies primarily on operation excision, but even if tumour is cut off completely, also has many patients to die of swollen
The recurrence and transfer of tumor, and traditional radiation and chemotherapy is also without apparent curative effect.Targeted drug especially tyrosine kinase presses down
So that the change of essence has occurred in GIST treatment, the treatment now for GIST has developed into needle for the appearance of preparation Imatinib
To the different state of an illness, the personalized complex treatment including operation, adjuvant treatment and lower rectal cancer is taken.Clinical treatment at present
A line targeted drug of GIST is Gleevec (Imatinib, Novartis), Second line Drug sotan (Sutent, Pfizer's system
Medicine), each patient year consumption is more than 10 ten thousand RMB.
But the curative effect of targeted drug with have GIST patient whether there is or not gene mutation and mutational site are closely related, different genes
The benefit of mutation type patient, adjuvant treatment have differences, therefore need to carry out genetic test before targeted therapy, to predict to target
The curative effect for the treatment of and guides the selection of targeted drug type and dosage.Its therapeutic regimen standardized comes into " between Chinese stomach and intestine
Matter tumor diagnoses and treatment common recognition " (version in 2013).
GIST patient can be divided into 3 classes: C-Kit saltant type (80~85%) according to its gene mutation type from molecular level,
PDGFR α saltant type (5~10%) and wild type GIST (10%).Wherein, C-Kit gene is located at human chromosome 4q12-13, entirely
Long 5230bp contains 21 exons.The mutant form multiplicity of C-Kit gene, including deletion mutation, point mutation and insertion mutation,
Mutational site occurs mainly in No. 9 (10%), No. 11 (70%), No. 13 (1%), 17 exons (1%).
Studies have shown that C-Kit gene mutation can not only assist the GIST that clarifies a diagnosis, but also its mutational site and GIST are targeted
Therapeutic response, the dosage of drug are related with prognosis.Wherein, from treatment with imatinib reaction, C-Kit gene has mutation
GIST case it is better than C-Kit wild type case therapeutic response effect, patient tumors are long without the life cycle being obviously in progress；C-Kit
Have in the GIST case of mutation, the curative effect of Imatinib is from high to low successively are as follows: 11 exon mutation persons, 9 exons are prominent
Change person, 13 exon mutation persons, 17 exon mutation persons；In addition, also can in Imatinib starting dosage selection
It is different because of mutational site difference.From tyrosine kinase inhibitor Sutent therapeutic response, Sutent second line treatment is former
The curative effect for sending out the mutation of 9 exon of C-Kit and wild type GIST case is mutated patient better than 11 exons；It treats secondary
The curative effect of the 13 exons mutation patient of C-Kit is better than secondary No. 17 mutation exon.Therefore, the prominent of C-Kit gene is detected
Point is conjugated, for instructing the rational use of medicines of GIST patient, to carry out GIST personalized medicines, there is important references value.
But the mutation of detection 13 exon of mankind C-Kit gene is surveyed mainly by traditional sequencing mode at present
Consuming time is long and at high cost for sequence, does not utilize common clinical detection.Even if there is a small number of reports for using fluorescence quantitative PCR method
Road, and only detecting to wherein some site of 13 exons, detection site is single, can not to 13 exons into
The assessment in the multiple mutational sites of row, is not able to satisfy the needs of practical application.
Summary of the invention
The purpose of the present invention is to provide a kind of kit of new quantitative PCR detection mankind's C-Kit gene mutation and sides
Method.
The information of 13 exon of C-Kit gene mutation is as follows:
The present invention provides it is a kind of detect mankind c-kit gene mutation type kit, it include SEQ ID NO.1~
Probe shown in primer pair shown in 4 and SEQ ID NO.5.
Wherein, it further includes Quality Control primer and Quality Control probe: primer pair shown in NO.6~8 SEQ ID and SEQ ID NO.9
Shown in probe.
The present invention also provides mentioned reagent boxes in the reagent of preparation detection 13 exon of mankind c-kit gene mutation
Purposes.
Wherein, 13 exon sports K642E, V654A or N655K mutation.
The present invention also provides primer pairs shown in NO.1~4 SEQ ID.
The present invention also provides probes shown in SEQ ID NO.5.
The present invention also provides a kind of methods of detection 13 exon of mankind c-kit gene mutation, it includes following step
It is rapid:
(1) it extracts sample DNA: taking tissue to be checked, extract DNA therein；
(2) gene magnification: the DNA in sample to be examined is expanded with mentioned reagent box；
(3) result detects: detecting to DNA cloning result.
Wherein, 13 exon sports K642E, V654A or N655K mutation.
The specific primer that the present invention designs, can be used for detecting 13 exon of C-Kit gene K642E, V654A and
N655K mutation.As long as detection kit of the present invention and method have measured mutation, so that it may confirm that 13 extras of C-Kit gene are aobvious
There are at least one mutation of tri- kinds of mutation types of K642E, V654A and N655K for son.So as to predict GIST patient to targeting
The therapeutic response of drug.
Kit provided by the invention can accurately detect 13 extras whether crowd to be checked C-Kit gene occurs simultaneously to be shown
3 mutational sites on son, more can accurate judgement GIST patient to be checked drug response, provide finger for the medication of GIST patient
It leads, and high sensitivity, specificity is good, and detects quick, simplicity, and it is low in cost, it can be used with large-scale popularization, before clinical application
Scape is good.
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The specific embodiment of form by the following examples remakes further specifically above content of the invention
It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on above content of the present invention
The technology realized all belongs to the scope of the present invention.
Detailed description of the invention
Fig. 1 is saltant type sample sequencing result figure
Fig. 2 is specific detection result figure
Fig. 3 is sensitivity technique result figure: number 1-4 is respectively that concentration is 8ng/ul, 4ng/ul, 2ng/ul, 1ng/ul
DNA cloning result.
Specific embodiment
It is described further below with embodiment, but the present invention is not limited to these embodiments.
Experiment reagent used in the present invention and instrument are as follows:
FastStartTaq DNA Polymerase: Roche company, Cat No.12 032 937 001 are purchased from；
Uracil DNA Glycosylase (UNG), heat labile 200U 2u/l: being purchased from Dalian Takara company,
Cat No.2820；
dU plus dNTP Mixture(12.5×)(dATP 2.5mM,dGTP 2.5mM,dCTP 2.5mM,dUTP
7.5mM): it is purchased from Dalian Takara company, Cat No.4035.
The kit and detection method of 1 present invention detection 13 exon of C-Kit gene mutation of embodiment
One, the composition of kit of the present invention
PCR amplification reagent (1 person-portion):
| Water | 14.85μl |
| 10XPCR buffer | 2.5μl |
| MgCl2(25mM) | 0.75μl |
| UTP PLUS | 0.5μl |
| Primer solution | 1μl |
| UNG | 0.2μl |
| Taq | 0.2μl |
| Total system | 20μl |
Wherein " Primer solution " is formulated as follows:
| C13F1(100nM/ml) | 3.125μl |
| C13F2(100nM/ml) | 3.125μl |
| C13F3(100nM/ml) | 3.125μl |
| C13R(100nM/ml) | 3.125μl |
| C13P(100nM/ml) | 0.625μl |
| H2O | 11.875μl |
| total | 25μl |
The amplimer and detection probe of 13 exon of c-kit gene mutation:
External control primer and probe:
Two, using kit of the present invention detection 13 exon of C-Kit gene mutation
1, sample DNA extracts: the DNA extraction kits such as Qiagen company, Tiangeng company, Promega company can be used
It extracts.
With paraffin organization sample, for Qiagen company kit, DNA is extracted.
1) the useless paraffin of organizational boundary is taken out using scalpel；
2) paraffin-embedded tissue is cut into the thin slice of 4 μ m-thicks；
3) take 2-6 pieces to be fitted into DNase/RNase Free EP pipe with sterilizing pincet rapidly (every is spread out area 500
(maximum) mm2, i.e. the square size of side length 1.6cm；
4) 800 μ l dimethylbenzene are added, 10s is shaken on oscillator, maximum revolution is centrifuged 3 minutes；
5) 800 μ l dimethylbenzene are added, 10s is shaken on oscillator, maximum revolution is centrifuged 3 minutes；
6) dimethylbenzene is abandoned, 800 μ l dehydrated alcohols are added, maximum revolution is centrifuged 3 minutes；
7) dehydrated alcohol is abandoned, sample is volatilized；
8) be added 180 μ l ALT buffer and 20 μ l proteinase K, 56 DEG C one hour or more, until limpid；
9) 90 DEG C one hour；
10) it is centrifuged 6000g 1min, takes supernatant；(this step is to prevent precipitate occlusion pillar)
11) 200 μ l AL are added, mix, 200 μ l ethyl alcohol are added, then mix；
12) simple centrifugation cleaning tube wall；
13) whole mixed liquors are added to DNA splitter, close lid, be centrifuged 6000g 1min, the collecting pipe renewed；
14) 500 μ l AW1,6000g are added and are centrifuged 1min；Change collecting pipe；
15) 500 μ l AW2,6000g centrifugation 1min are added, change collecting pipe；
16) it is centrifuged 3min, dry pillar at full speed；
17) a clean 1.5ml collecting pipe is changed, prepares to collect DNA；20-30 μ l ATE is added, keeps 1min in room temperature
With dissolving DNA.Centrifugation 1min collects DNA at full speed.
2, quantitative pcr amplification
The amplimer and detection probe of c-kit gene mutation:
C13 primer solution (Primer solution) is formulated as follows, and after making primer solution, is added in quantitative PCR system
1ul.
| C13F1 | 3.125μl |
| C13F2 | 3.125μl |
| C13F3 | 3.125μl |
| C13R | 3.125μl |
| C13P | 0.625μl |
| H2O | 11.875μl |
| total | 25μl |
C-kit external control primer solution (Primer solution) is formulated as follows, after making primer solution, in quantitative PCR system
Middle addition 1ul.
| 471 is positive | 3.125ml |
| 801 is positive | 3.125ml |
| C11R | 3.125ml |
| C11P | 0.625ml |
| H2O | 15ml |
| Total | 25ml |
1) quantitative PCR system is prepared:
It is loaded according to following table into 2 PCR pipes:
| Guan Hao | Detect mutation type | Mutant designations |
| 1 | c-kit13 | K642E, V654A and N655K |
| 2 | C-kit external control |
2) BIO-RAD CFX96 machine PCR thermocycling program is arranged:
3, result interpretation:
Using external control signal as standard, signal Ct value shows the amount of loading DNA within allowed band in 15-25.It is all
The external control curve of experiment sample should rise, and otherwise extract DNA detection again.Hole to be checked (hole number 1) Ct value is read, Ct value is
0 (either without amplification curve) was greater than 29 interpretations feminine gender, was determined as no C13 mutation；Ct value is less than 28 interpretations telltale hole sun
Property.28~29 repeat to test, if being still mutated in 28~29 interpretation C13 weakly positives.
Wherein, positive, weakly positive: representative sample has mutation, mutation type K642E, V654A or N655K.
Illustrate beneficial effects of the present invention with the mode of experimental example below:
The accuracy of the kit of the present invention of experimental example 1 and method detects
1, sample to be examined
Choosing known mutations type is that (respectively K642E, V654A, N655K are prominent for the mutation of 13 exon of c-kit gene
Become) each 1 of clinical GIST paraffin organization sample (wherein, K642E mutation sequencing result see Fig. 1) and known wild type clinic
1, GIST paraffin organization sample detects mankind c-kit gene mutation type.
2, detection method
The method of the present invention: it using the kit of embodiment 1, is detected according to the method for embodiment 1.
Sample DNA extracting method: it is extracted with Qiagen company kit:
1) the useless paraffin of organizational boundary is taken out using scalpel；
2) paraffin-embedded tissue is cut into the thin slice of 4 μ m-thicks；
3) take 2-6 pieces to be fitted into DNase/RNase Free EP pipe with sterilizing pincet rapidly (every is spread out area 500
(maximum) mm2, i.e. the square size of side length 1.6cm；
4) 800 μ l dimethylbenzene are added, 10s is shaken on oscillator, maximum revolution is centrifuged 3 minutes；
5) 800 μ l dimethylbenzene are added, 10s is shaken on oscillator, maximum revolution is centrifuged 3 minutes；
6) dimethylbenzene is abandoned, 800 μ l dehydrated alcohols are added, maximum revolution is centrifuged 3 minutes；
7) dehydrated alcohol is abandoned, sample is volatilized；
8) be added 180 μ l ALT buffer and 20 μ l proteinase K, 56 DEG C one hour or more, until limpid；
9) 90 DEG C one hour；
10) it is centrifuged 6000g 1min, takes supernatant；(this step is to prevent precipitate occlusion pillar)
11) 200 μ l AL are added, mix, 200 μ l ethyl alcohol are added, then mix；
12) simple centrifugation cleaning tube wall；
13) whole mixed liquors are added to DNA splitter, close lid, be centrifuged 6000g 1min, the collecting pipe renewed；
14) 500 μ l AW1,6000g are added and are centrifuged 1min；Change collecting pipe；
15) 500 μ l AW2,6000g centrifugation 1min are added, change collecting pipe；
16) it is centrifuged 3min, dry pillar at full speed；
17) a clean 1.5ml collecting pipe is changed, prepares to collect DNA；20-30 μ l ATE is added, keeps 1min in room temperature
With dissolving DNA.Centrifugation 1min collects DNA at full speed.
3, experimental result
If saltant type sample signal Ct value is within 28, then it represents that the inspection of 13 exon of c-kit gene occurs in sample
Survey the mutation of at least one of mutation type (K642E, V654A and N655K mutation).
3 saltant type sample signal Ct values of the invention are within 28, the wherein sequencing result of K642E saltant type sample
As shown in Figure 2.In Fig. 2, curve 1 is saltant type sample signal；Curve 2 is external control signal, and curve 3 is negative control (wild type
Control) signal, within 28, K642E's sample signal Ct value for indicating that 13 exon of c-kit gene occurs in the sample dashes forward
Become.
The experiment results show that the method for the present invention and the testing result of kit are compared with known testing result: result is consistent,
Illustrate the method for the present invention and kit can accurately the 13 exon mutation of c-kit gene, accuracy be strong；In addition, negative control
Sample Ct value is greater than 30, for feminine gender, illustrates that the specificity of the method for the present invention and kit is good.
The sensitivity analysis of the kit of the present invention of experimental example 2 and method
1, test method
Take the DNA containing the mutation of 13 exon of mankind c-kit gene, quantitative to concentration is 8ng/ul, is carried out etc. than dilute
It releases, the concentration after dilution is respectively 8ng/ul, 4ng/ul, 2ng/ul, 1ng/ul, 0.5ng/ul
With the kit of embodiment 1, detected according to the quantitative pcr amplification of embodiment 1.
2, experimental result
As shown in figure 3, kit of the present invention can detecte the low concentration sample that concentration is 2ng/ul, high sensitivity.
To sum up, kit provided by the invention can accurately detect the mutation of 13 exon of mankind C-Kit gene, specificity
And high sensitivity, detection is quick, easy, and it is low in cost, it can be used with large-scale popularization, potential applicability in clinical practice is good.
Claims (4)
1. a kind of kit for detecting mankind c-kit gene mutation type, it is characterised in that: it includes SEQ ID NO.1~4 institute
Probe shown in the primer pair and SEQ ID NO.5 shown.
2. kit according to claim 1, it is characterised in that: it further includes Quality Control primer and Quality Control probe: SEQ ID
Primer pair shown in NO.6~8 and probe shown in SEQ ID NO.9.
3. kit of any of claims 1 or 2 is in the reagent of preparation detection 13 exon of mankind c-kit gene mutation
Purposes.
4. purposes according to claim 3, it is characterised in that: 13 exon sport K642E, V654A or
N655K mutation.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104328184A (en) * | 2014-10-30 | 2015-02-04 | 武汉百泰基因工程有限公司 | Primer, probe, locked nucleic acid probe, kit and detection method for detecting C-kit gene mutation |
| CN104561250A (en) * | 2013-10-22 | 2015-04-29 | 联合基因生物医药有限公司 | Method and primer for detecting imatinib targeted medication gene |
| CN105039534A (en) * | 2015-07-08 | 2015-11-11 | 广州和实生物技术有限公司 | C-KIT gene multipoint mutation single tube fast detection method and kit |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104561250A (en) * | 2013-10-22 | 2015-04-29 | 联合基因生物医药有限公司 | Method and primer for detecting imatinib targeted medication gene |
| CN104328184A (en) * | 2014-10-30 | 2015-02-04 | 武汉百泰基因工程有限公司 | Primer, probe, locked nucleic acid probe, kit and detection method for detecting C-kit gene mutation |
| CN105039534A (en) * | 2015-07-08 | 2015-11-11 | 广州和实生物技术有限公司 | C-KIT gene multipoint mutation single tube fast detection method and kit |
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