CN106011254A - Diagnostic Kit and method for detecting mutation of exon 17 of human C-Kit gene - Google Patents
Diagnostic Kit and method for detecting mutation of exon 17 of human C-Kit gene Download PDFInfo
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- CN106011254A CN106011254A CN201610440458.8A CN201610440458A CN106011254A CN 106011254 A CN106011254 A CN 106011254A CN 201610440458 A CN201610440458 A CN 201610440458A CN 106011254 A CN106011254 A CN 106011254A
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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 17 of the human c-kit gene. The kit and the method can accurately detect the D816V, D820H, N822I and N822K mutations of the exon 17 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 field of gene detection, be specifically related to a kind of detect the mankind's C-Kit gene 17 exon sudden change
Diagnostic kit and method.
Background technology
Gastrointestinal stromal tumor (gastrointestinal stromaltumors, GIST) is that a class originates between gastrointestinal tract
The tumor of leaf texture, is modal leaf source property tumor of gastrointestinal tract, is mainly in middle-older patient, and men and women's sickness rate is without the poorest
Different.Mazur etc. nineteen eighty-three propose, GIST be class one zone not in the independent tumors type of leiomyoma and neurogenic tumour,
Its direct cause of disease is the acquired function mutation of oncogene.
Early treatment GIST relies primarily on excision, but even if tumor is excised completely, also has a lot of patient to die from swollen
The recurrence of tumor and transfer, and traditional radiation and chemotherapy does not has obvious curative effect yet.Targeted drug particularly tyrosine kinase presses down
The appearance of preparation imatinib makes GIST treatment there occurs the change of essence, and the treatment now for GIST has developed into pin
To the different state of an illness, take to include operation, auxiliary treatment and the personalized Comprehensive Treatment of lower rectal cancer.Clinical treatment at present
The one line targeted drug of GIST is imatinib mesylate (imatinib, Novartis), Second line Drug SU11248 (Sutent, Pfizer's system
Medicine), consume each patient's year and be more than 10 ten thousand RMB.
But the gene that the curative effect of targeted drug is closely related, different with or without gene mutation and mutational site with there being GIST patient
Mutation type patient, the benefit of auxiliary treatment there are differences, and therefore needs before targeted therapy to carry out gene test, to predict targeting
The curative effect for the treatment of and guides the selection of targeted drug kind and dosage.The therapeutic regimen of its specification comes into " between China's gastrointestinal
Matter tumor diagnoses and treatment is known together " (version in 2013).
GIST patient, according to its gene mutation type, can be divided into 3 classes from molecular level: C-Kit saltant type (80～85%),
PDGFR α saltant type (5～10%) and wild type GIST (10%).Wherein, C-Kit gene is positioned at human chromosome 4q12-13, entirely
Long 5230bp, containing 21 exons.The mutant form of C-Kit gene is various, 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%).
Research shows, C-Kit gene mutation not only can assist the GIST that clarifies a diagnosis, and its mutational site and GIST targeting
The therapeutic response of medicine, dosage are relevant with prognosis.Wherein, from treatment with imatinib reaction, C-Kit gene has sudden change
GIST case more effective than C-Kit wild type case therapeutic response, patient tumors is long without the life cycle of substantially progress；C-Kit
Having in the GIST case of sudden change, the curative effect of imatinib is followed successively by from high to low: 11 exon sudden change persons, 9 exons are dashed forward
Change person, 13 exon sudden change persons, 17 exon sudden change persons；Select it addition, initiate dosage at imatinib, also can
It is different because mutational site is different.From tyrosine kinase inhibitor Sutent therapeutic response, Sutent second line treatment is former
The curative effect sending out the sudden change of C-Kit 9 exon and wild type GIST case is better than 11 exon sudden change patients；Treatment Secondary cases
The curative effect of the 13 exon sudden change patients of C-Kit is better than secondary 17 sudden change exon.Therefore, detection C-Kit gene is prominent
Displacement point, for instructing the rational use of drug of GIST patient, to carry out GIST personalized medicines, has important references and is worth.
But, detection mankind's C-Kit gene 17 exon sudden change at present, mainly by traditional order-checking mode, survey
The consuming time length of sequence and cost are high, do not utilize common Clinical detection.Even if there being minority to use the report of fluorescence quantitative PCR method
Road, is also only to detect wherein some site of 17 exons, and detection site is single, it is impossible to enter 17 exons
The assessment in the multiple mutational sites of row, it is impossible to meet the needs of reality application.
Summary of the invention
It is an object of the invention to provide test kit and the side of a kind of new quantitative PCR detection mankind's C-Kit gene mutation
Method.
The information of C-Kit gene 17 exon sudden change is as follows:
The invention provides a kind of test kit detecting mankind's c-kit gene mutation type, it include SEQ ID NO.1～
Primer shown in 4 to and SEQ ID NO.5 shown in probe.
Wherein, it also includes Quality Control primer and Quality Control probe: primer pair shown in SEQ ID NO.6～8 and SEQ ID NO.9
Shown probe.
Present invention also offers described test kit in the reagent of preparation detection mankind's c-kit gene 17 exon sudden change
Purposes.
Wherein, described 17 exons sport D816V, D820H, N822I or N822K sudden change.
Present invention also offers primer pair shown in SEQ ID NO.1～4.
Present invention also offers the probe shown in SEQ ID NO.5.
Present invention also offers a kind of method detecting the sudden change of mankind's c-kit gene 17 exon, it includes walking as follows
Rapid:
(1) sample DNA is extracted: take tissue to be checked, extract DNA therein；
(2) gene amplification: the DNA treated in sample basis with above-mentioned test kit expands；
(3) result detection: DNA cloning result is detected.
Wherein, described 17 exons sport D816V, D820H, N822I or N822K sudden change.
The present invention design specific primer, may be used for detect the D816V of C-Kit gene 17 exon, D820H,
N822I and N822K suddenlys change.As long as detection kit of the present invention and method have measured sudden change, it is possible to confirm the 17 of C-Kit gene
There is at least one sudden change of tetra-kinds of mutation types of D816V, D820H, N822I and N822K in exon.Such that it is able to prediction
GIST patient's therapeutic response to targeted drug.
The test kit that the present invention provides can accurately detect whether crowd to be checked occurs that 17 extras of C-Kit gene show simultaneously
4 mutational sites on son, more can accurately judge the drug reaction of GIST patient to be checked, and the medication offer for GIST patient refers to
Lead, and highly sensitive, specificity is good, and detects quick, easy, with low cost, can use with large-scale popularization, before clinical practice
Scape is good.
Obviously, according to the foregoing of the present invention, according to ordinary technical knowledge and the customary means of this area, without departing from
Under the present invention above-mentioned basic fundamental thought premise, it is also possible to make the amendment of other various ways, replace or change.
The detailed description of the invention of form by the following examples, remakes the most specifically the foregoing of the present invention
Bright.But this should not being interpreted as, the scope of the above-mentioned theme of the present invention is only limitted to Examples below.All based on foregoing of the present invention
The technology realized belongs to the scope of the present invention.
Accompanying drawing explanation
Fig. 1 is saltant type sample sequencing result figure
Fig. 2 is specific detection result figure
Fig. 3 is sensitivity technique result figure: numbering 1-5 respectively concentration is 8ng/ul, 4ng/ul, 2ng/ul, 1ng/ul,
The DNA cloning result of 0.5ng/ul.
Detailed description of the invention
It is described further with embodiment below, but the present invention is not limited to these embodiments.
Experiment reagent used by the present invention is as follows with instrument:
FastStartTaq DNA Polymerase: purchased from Roche company, Cat No.12 032 937 001；
Uracil DNA Glycosylase (UNG), heat labile 200U 2u/l: 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): purchased from Dalian Takara company, Cat No.4035.
Embodiment 1 present invention detects test kit and the detection method of C-Kit gene 17 exon sudden change
One, the composition of test kit of the present invention
PCR amplifing 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:
| C17F(100nM/ml) | 12.5μl |
| C17F2(100nM/ml) | 12.5μl |
| C17F3(100nM/ml) | 12.5μl |
| C17R(100nM/ml) | 12.5μl |
| C17P(100nM/ml) | 2.5μl |
| H2O | 47.5μl |
| total | 100μl |
The amplimer of c-kit gene 17 exon sudden change and detection probe:
External control primer and probe:
Two, test kit of the present invention detection C-Kit gene 17 exon sudden change is used
1, sample DNA extracts: can use the DNA extraction kit such as Qiagen company, Tian Gen company, Promega company
Extract.
With paraffin organization sample, as a example by Qiagen company test kit, extract DNA.
1) scalpel is utilized to take out the paraffin that organizational boundary is useless；
2) paraffin-embedded tissue is cut into the thin slice of 4 μ m-thick；
3) rapid taking in 26 pieces of loading DNase/RNase Free EP pipes with sterilizing pincet (spreads area 500 out for every
(maximum) mm2, i.e. the square size of length of side 1.6cm；
4) adding 800 μ l dimethylbenzene, agitator shakes 10s, maximum revolution is centrifuged 3 minutes；
5) adding 800 μ l dimethylbenzene, agitator shakes 10s, maximum revolution is centrifuged 3 minutes；
6) abandoning dimethylbenzene, add 800 μ l dehydrated alcohol, maximum revolution is centrifuged 3 minutes；
7) abandon dehydrated alcohol, volatilize sample；
8) add 180 μ l ALT buffer and 20 μ l proteinase K, 56 DEG C more than one hour, until limpid；
9) 90 DEG C one hour；
10) centrifugal 6000g 1min, takes supernatant；(this step is to prevent precipitate occlusion pillar)
11) 200 μ l AL are added, mixing, add 200 μ l ethanol, then mix；
12) simple centrifugal cleaning tube wall；
13) whole mixed liquors are joined DNA detached dowel, close lid, centrifugal 6000g 1min, the collecting pipe renewed；
14) add 500 μ l AW1,6000g and be centrifuged 1min；Change collecting pipe；
15) adding 500 μ l AW2,6000g is centrifuged 1min, changes collecting pipe；
16) the most centrifugal 3min, is dried pillar；
17) change a clean 1.5ml collecting pipe, prepare to collect DNA；Add 20-30 μ l ATE, keep 1min in room temperature
With dissolving DNA.The most centrifugal 1min collects DNA.
2, quantitative pcr amplification
The amplimer of c-kit gene mutation and detection probe:
C17 primer solution (Primer solution) is formulated as follows, and after making primer solution, adds in quantitative PCR system
1ul.
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 preparation:
| Water | 14.85μl |
| 10XPCR buffer | 2.5μl |
| MgCl2 | 0.75μl |
| UTP PLUS | 0.5μl |
| Primer solution | 1μl |
| UNG | 0.2μl |
| Taq | 0.2μl |
| DNA | 5 μ l (concentration is 3ng/ μ l) |
| Total system | 25μl |
It is loaded into 2 PCR pipe according to following form:
| Guan Hao | Detection mutation type | Mutant designations |
| 1 | c-kit17 | D816V, D820H, N822I and N822K |
| 2 | C-kit external control |
2) BIO-RAD CFX96 machine PCR thermocycling program is arranged:
3, result interpretation:
With external control signal as standard, its signal Ct value, at 15-25, shows that the amount of loading DNA is within allowed band.All
The external control curve of experiment sample all should rise, and the most again extracts DNA detection.Reading hole to be checked (hole number 1) Ct value, Ct value is
0 (or without amplification curve) or negative more than 29 interpretations, it is determined that for without C17 sudden change；Ct value is less than this telltale hole sun of 28 interpretations
Property.28～29 repeat experiment, if still at the 28～29 weak Positive mutants of interpretation C17.
Wherein, the positive positive, weak: representative sample has sudden change, mutation type is D816V, D820H, N822I or N822K.
By the mode of experimental example beneficial effects of the present invention is described below:
Experimental example 1 test kit of the present invention and the accuracy of method and specific detection
1, sample to be checked
Choose known mutations type be c-kit gene 17 exon sudden change (respectively D816V, D820H, N822I,
N822K suddenlys change) clinical each 1 example of GIST paraffin organization sample (wherein, the sequencing result of D816V sudden change is shown in Fig. 1) and known open country
Raw type clinic GIST paraffin organization sample 1 example, detects mankind's c-kit gene mutation type.
2, detection method
The inventive method: use the test kit of embodiment 1, detect according to the method for embodiment 1.
Sample DNA extracting method: extract with Qiagen company test kit:
1) scalpel is utilized to take out the paraffin that organizational boundary is useless；
2) paraffin-embedded tissue is cut into the thin slice of 4 μ m-thick；
3) rapid taking in 26 pieces of loading DNase/RNase Free EP pipes with sterilizing pincet (spreads area 500 out for every
(maximum) mm2, i.e. the square size of length of side 1.6cm；
4) adding 800 μ l dimethylbenzene, agitator shakes 10s, maximum revolution is centrifuged 3 minutes；
5) adding 800 μ l dimethylbenzene, agitator shakes 10s, maximum revolution is centrifuged 3 minutes；
6) abandoning dimethylbenzene, add 800 μ l dehydrated alcohol, maximum revolution is centrifuged 3 minutes；
7) abandon dehydrated alcohol, volatilize sample；
8) add 180 μ l ALT buffer and 20 μ l proteinase K, 56 DEG C more than one hour, until limpid；
9) 90 DEG C one hour；
10) centrifugal 6000g 1min, takes supernatant；(this step is to prevent precipitate occlusion pillar)
11) 200 μ l AL are added, mixing, add 200 μ l ethanol, then mix；
12) simple centrifugal cleaning tube wall；
13) whole mixed liquors are joined DNA detached dowel, close lid, centrifugal 6000g 1min, the collecting pipe renewed；
14) add 500 μ l AW1,6000g and be centrifuged 1min；Change collecting pipe；
15) adding 500 μ l AW2,6000g is centrifuged 1min, changes collecting pipe；
16) the most centrifugal 3min, is dried pillar；
17) change a clean 1.5ml collecting pipe, prepare to collect DNA；Add 20-30 μ l ATE, keep 1min in room temperature
With dissolving DNA.The most centrifugal 1min collects DNA.
3, experimental result
If saltant type sample signal Ct value is within 28, then it represents that sample occurs in that c-kit gene 17 exon is examined
Survey at least one sudden change in mutation type (D816V, D820H, N822I and N822K suddenly change).
The 4 example saltant type sample signal Ct values of the present invention all within 28, the wherein sequencing result of D816V 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
Comparison) signal, sample signal Ct value, within 28, represents that this sample occurs in that the D816V of c-kit gene 17 exon dashes forward
Become.
Experimental result illustrates, the inventive method compares with known testing result with the testing result of test kit: result is consistent,
Illustrating that the inventive method and test kit can accurately suddenly change by c-kit gene 17 exon, accuracy is strong；It addition, negative control
Sample Ct value is more than 30, for feminine gender, illustrates that the specificity of the inventive method and test kit is good.
Experimental example 2 test kit of the present invention and the sensitive analysis of method
1, test method
Take the DNA containing the sudden change of mankind's c-kit gene 17 exon, be quantitatively 8ng/ul to concentration, carry out geometric ratio dilute
Releasing, the concentration after dilution is respectively 8ng/ul, 4ng/ul, 2ng/ul, 1ng/ul, 0.5ng/ul.
With the test kit of embodiment 1, detect according to the quantitative pcr amplification of embodiment 1.
2, experimental result
As it is shown on figure 3, test kit of the present invention can detect the low concentration sample that concentration is 1ng/ul, highly sensitive.
To sum up, the test kit that the present invention provides can accurately detect the sudden change of mankind's C-Kit gene 17 exon, and detection is fast
Fast, easy, with low cost, can use with large-scale popularization, potential applicability in clinical practice is good.
Claims (8)
1. the test kit detecting mankind's c-kit gene mutation type, it is characterised in that: it includes SEQ ID NO.1～4 institute
The primer shown to and SEQ ID NO.5 shown in probe.
Test kit the most according to claim 1, it is characterised in that: it also includes Quality Control primer and Quality Control probe: SEQ ID
Primer pair shown in NO.6～8 and the probe shown in SEQ ID NO.9.
3. the test kit described in claim 1 or 2 is in the reagent of preparation detection mankind's c-kit gene 17 exon sudden change
Purposes.
Purposes the most according to claim 3, it is characterised in that: described 17 exons sport D816V, D820H,
N822I or N822K suddenlys change.
Primer pair shown in 5.SEQ ID NO.1～4.
Probe shown in 6.SEQ ID NO.5.
7. the method detecting the sudden change of mankind's c-kit gene 17 exon, it is characterised in that: it comprises the steps:
(1) sample DNA is extracted: take tissue to be checked, extract DNA therein；
(2) gene amplification: the DNA treated in sample basis with the test kit described in claim 1 or 2 expands；
(3) result detection: DNA cloning result is detected.
Method the most according to claim 7, it is characterised in that: described 17 exons sport D816V, D820H,
N822I or N822K suddenlys change.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2674338C1 (en) * | 2017-10-09 | 2018-12-07 | Федеральное Государственное Бюджетное Учреждение Науки Институт Молекулярной Биологии Им. В.А. Энгельгардта Российской Академии Наук (Имб Ран) | Method for analysis of somatic mutations in braf, nras and kit genes using lna-blocking multiplex pcr and subsequent hybridization with oligonucleotide biological microchip (biochip) |
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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 |
| 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 |
| CN105349682A (en) * | 2015-12-07 | 2016-02-24 | 湖南圣维基因科技有限公司 | C-KIT gene D816V mutation fluorescent PCR detection kit |
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- 2016-06-17 CN CN201610440458.8A patent/CN106011254A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN105349682A (en) * | 2015-12-07 | 2016-02-24 | 湖南圣维基因科技有限公司 | C-KIT gene D816V mutation fluorescent PCR detection kit |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2674338C1 (en) * | 2017-10-09 | 2018-12-07 | Федеральное Государственное Бюджетное Учреждение Науки Институт Молекулярной Биологии Им. В.А. Энгельгардта Российской Академии Наук (Имб Ран) | Method for analysis of somatic mutations in braf, nras and kit genes using lna-blocking multiplex pcr and subsequent hybridization with oligonucleotide biological microchip (biochip) |
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