CN105112500B - A kind of primer, probe and kit for being used to detect C-KIT gene mutations - Google Patents

A kind of primer, probe and kit for being used to detect C-KIT gene mutations Download PDF

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CN105112500B
CN105112500B CN201510364301.7A CN201510364301A CN105112500B CN 105112500 B CN105112500 B CN 105112500B CN 201510364301 A CN201510364301 A CN 201510364301A CN 105112500 B CN105112500 B CN 105112500B
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kit
probe
primer
gene
detection
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CN105112500A (en
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莫敏俐
李晖
陈钊
丁凤
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YAKANGBO BIOLOGICAL SCIENCE AND TECHNOLOGY Co Ltd BEIJING
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YAKANGBO BIOLOGICAL SCIENCE AND TECHNOLOGY Co Ltd BEIJING
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Abstract

The invention discloses a kind of for detecting the primer, probe and kit of C KIT gene mutations.The primer is as shown in SEQ ID No.1 SEQ ID No.2, and the probe is as shown in SEQ ID No.3.Real-time fluorescence PCR is carried out using the kit of the present invention, 23 1728 deletion mutations of C KIT gene 17s can be detected.

Description

A kind of primer, probe and kit for being used to detect C-KIT gene mutations
Technical field
The present invention relates to biotechnology, specifically, be related to it is a kind of for detect C-KIT gene mutations primer, Probe and related kit.
Background technology
C-KIT is a kind of proto-oncogene, positioned at chromosome 4q11-12, overall length about 80kb.The C-KIT albumen of C-KIT codings Belong to type III receptor tyrosine kinase family member, it is the transmembrane protein that molecular weight is about 145KD, contains the junket of intracellular Histidine kinase area, transmembrane region and ligand binding site extracellular region.Itself tyrosine protein kinase can be activated after ligand is in connection Enzymatic activity activates downstream signal transduction access, so as to adjust the growth of cell and proliferation by series reaction.
Gastrointestinal stromal tumor (GIST) is most common leaf source property tumour of alimentary canal.Research shows that in GIST patient C-KIT gene mutation rates are about 90%.
Sanger PCR sequencing PCRs are the main methods of C-KIT detection in Gene Mutation at present.However, the sensitivity of this method is low, It can lead to missing inspection and the generation of false negative, and detection time is long, the actual demand of clinical detection can not be met.It is clinically urgent It needs to develop a kind of highly sensitive, quick C-KIT detection in Gene Mutation technology.
The present inventor is found that have with gastrointestinal stromal tumor in the routine testing of gastrointestinal stromal tumor (GIST) Close positioned at the new mutation site of C-KIT genes, i.e. 1723-1728 deletion mutation.The present inventor is directed to the mutation A kind of detection kit quick, highly sensitive, easy to operate and relevant detection method are developed, it is pre- to can be used for GIST chemotherapy Afterwards.
Invention content
The purpose of the present invention is to provide a kind of for detecting the primer and probe of C-KIT gene mutations, the mutation is 1723-1728 6 base mutations of missing (1723_1728 del 6, Q575_L576 del).The primer of the present invention and probe packet Include following sequence：
Forward primer (F)：GTTTACATAGACCCAACACCTTA(SEQ ID No：1)
Reverse primer (R)：CCCTGTTTCATACTGACCAA(SEQ ID No：2)
Probe (PB)：CTCAGCCTGTTTCTGGGAAACTCCC(SEQ ID No：3)
Another aspect of the present invention provides a kind of detection kit for being used to detect C-KIT gene mutations, the kit packet Include the primer shown in SEQ ID No.1-SEQ ID No.2 and the probe shown in SEQ ID No.3.
According to used PCR method, kit of the invention can also include reference gene and internal control gene.
Another aspect of the present invention provides one kind for detecting C-KIT gene mutation methods, includes the following steps：
(1) for the mutational site of C-KIT, specific primer and probe are designed；
(2) genomic DNA in extraction detection sample, detection sample include fresh pathological tissue, paraffin-embedded tissue, whole blood With blood plasma etc.；
(3) real-time fluorescent PCR amplification reaction system is established；
(4) the Ct values shown according to fluorescent PCR instrument judge testing result：The FAM fluorescence intensities of reaction system are detected, with FAM reaches during the threshold value of setting required cycle-index Ct values as negative, Positive judgement standards, Ct values ＞ 38 or without amplification For feminine gender, Ct value≤38 are the positive.
Present invention employs specific primers and probe technique, can be with specific detection C-KIT gene mutations.The method spirit Sensitivity height, high specificity, detection speed are fast.
Description of the drawings
Fig. 1 is the PCR figures for detecting negative sample.
Fig. 2 is the PCR figures of detection mutation positive sample.
Specific embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments be only used for the present invention and It is not used in and limits the scope of the invention.Test method without specific conditions in embodiment, it is ripe according to those skilled in the art The normal condition known or the condition suggested according to manufacturer.
One, principles
After the specific primer of the present invention is combined with probe with DNA profiling, Taq archaeal dna polymerases are with deoxynucleotide (dNTP) it is substrate, reference gene (Internal Reference, IR) and C-KIT genic mutation type genes is expanded in vitro Increase.Detection hydrolyzes release fluorescence, the progress of monitoring PCR reactions determines C-KIT using Fluorescence PCR assay by specific probe Gene mutation situation.
Reference gene detection architecture has been separately provided in the kit of the present invention.Reference gene is to be different from C-KIT bases to be checked The house-keeping gene of cause.By detecting the amplification situation (FAM channels) of reference gene, it can analyze whether DNA to be checked can normally be expanded Increase, so as to exclude the situation that DNA purity, concentration are bad or PCR detections are caused to fail containing PCR inhibitor etc..
There is provided internal control gene (Internal simultaneously in C-KIT genic mutation type detection architectures for the kit of the present invention Control, IC) detection architecture.Two kinds of systems are carried out at the same time reaction in same PCR pipe.Internal control gene be also be different from it is to be checked The house-keeping gene of gene C-KIT.It is FAM fluorophors by the probe modification for identifying C-KIT gene mutation templates, and in identifying The probe modification for controlling gene template is HEX fluorophors.By detecting internal control gene magnification situation (HEX channels), it can analyze and treat Whether inspection DNA can normally be expanded, and contains PCR inhibitor etc. so as to exclude leakage reagent adding or sample, sample and PCR detections is caused to lose Situation about losing.
When carrying out the judgement of this kit testing result, FAM, HEX Air conduct measurement should all be without amplifications in negative quality-control product (NC) (not in typical S types curve.Note：Typical S types curve shows as exponential phase, straight line phase and plateau successively) or without Ct values, FAM, HEX Air conduct measurement should have amplification (in typical S types curve) and Ct value≤28 in positive quality control product (PC)；Otherwise it is assumed that Experiment is invalid, need to repeat to test.
Two, experiment materials and equipment
1. for mutational site design synthesis specific primer and probe
Special primer and probe are designed for C-KIT gene mutation sites.By specific primer and probe optimization, so as to Realize highly sensitive and quick detection.
Primer and probe after optimization is as follows：
Forward primer (F)：GTTTACATAGACCCAACACCTTA(SEQ ID No：1)
Reverse primer (R)：CCCTGTTTCATACTGACCAA(SEQ ID No：2)
Probe (PB)：FAM-CTCAGCCTGTTTCTGGGAAACTCCC-BHQ-1(SEQ ID No：3, containing fluorescent marker)
2. detect sample process and the extraction of DNA
It can be fresh pathological tissue, paraffin-embedded tissue, whole blood, blood plasma and seroperitoneum to detect sample.Below only with It is illustrated for paraffin-embedded tissue sample.
Before sample collection, patient without tyrosine kinase inhibitor (tyrosine kinase inhibitors, TKIs) class drug therapy；Since DNA sample quality can influence testing result, the paraffin embedding in DNA sample source should determine that Contain cancerous tissue cell, and the 25% of no less than entire sample in tissue samples；And DNA sample OD260/OD280=1.8 ± 0.2, OD260/OD230 >=1.7；A concentration of 5-10ng/ μ l.
Conserving time limit is no more than 12 months paraffin-embedded tissue sample at room temperature, and the DNA sample after extraction is cold at -20 DEG C Conserving time limit is no more than 6 months under the conditions of jelly.
3. applicable instrument
Stratagene Mx3000P。
4. the composition of kit
Kit forms are shown in Table 1.Without nucleic acid extraction composition in kit, QIAamp DNA FFPE Tissue are used (Qiagen companies produce Kit, article No.：56404) kit completes the extraction of paraffin-embedded tissue sample DNA.Detection reagent forms And C-KIT genes mutational site to be checked is shown in Table 2.IR detection reagents are containing only reference gene detection architecture (FAM channels), C-KIT inspections Test agent contains C-KIT detection in gene mutation system (FAM channels) and internal control genetic test system (HEX channels) simultaneously.
Table 1：Kit forms
The particular sequence of wherein described reference gene and internal control gene can be by those skilled in the art according to experimental conditions It is readily determined.
Three, detection methods
1. QIAamp DNA FFPE Tissue Kit (production of Qiagen companies) kit is used, according to operation instructions Carry out paraffin-embedded tissue sample DNA extraction.
2. take the detection reagent and positive quality control product (PC) in kit.Test agent to be checked and positive quality control product PC melt Of short duration centrifugation afterwards, is placed on ice.
3. volume ratio mixing IR detection reagents and Taq archaeal dna polymerases by 17: 0.3, by 17: 0.3 volume ratio Mix C-KIT detection reagents and Taq archaeal dna polymerases.IR detection reagents and C-KIT detection reagents are dispensed to eight companies by 17.3/ hole Guan Zhong.2.7 μ l DNA samples to be checked, negative matter are separately added into eight connecting legs equipped with IR detection reagents and C-KIT detection reagents Control product (NC, the buffer solution of dissolving DNA, provided for oneself by user) and positive quality control product PC blow and beat mixing, cover tightly after pipe lid it is of short duration from The heart.Pay attention to：Vortex oscillation instrument is not used during mixing；Subsequent operation is carried out after mixing immediately.
4. the setting of fluorescent PCR instrument sense channel needs simultaneous selection FAM, HEX channel (reference dye is set as "None").Reaction Program setting is following (table 2)：
Table 2：
The explanation of four, testing results
1.Ct values determine：The baseline of Stratagene MX3000P fluorescent PCR instrument is set first：Selection " is suitble to baseline Fluorescence signal when (Adaptive baseline) " is set, threshold value (threshold) setting principle are just above with threshold line The peak of normal feminine gender quality-control product NC amplification curves (random noise line), even negative quality-control product NC amplification curves are shown Subject to " No Ct ".Ct value of each sample in each site primer is read from software.
2. qualitative analysis：
(1) quality of experiments is judged：If in NC FAM, HEX Air conduct measurement without amplification (not in typical S types curve.Note：Allusion quotation The S types curve of type shows as exponential phase, straight line phase and plateau successively) or without Ct values, FAM, HEX Air conduct measurement have expansion in PC Increase (being in typical S types curve) and Ct value≤28, then can continue to analyze；Otherwise it is assumed that experiment is invalid, need to repeat to test.
(2) reference gene (IR) detection case is judged in DNA sample to be checked：If reference gene detection (FAM channels) has expansion Increasing and 19≤Ct value≤25, then can continue to analyze；If its Ct value is smaller, then it is assumed that DNA sample excessive concentration；If its Ct value is larger Or without amplification, then it is assumed that DNA sample concentration is too low, degrade or may wherein contain PCR inhibitor.
(3) internal control genetic test situation is judged in DNA sample to be checked：If internal control genetic test (HEX channels) have amplification and Ct value≤25 can then continue to analyze；If internal control genetic test (HEX channels) Ct values are larger or without amplification, but mutational site is detected (FAM channels) has amplification and Ct value≤38, then can continue analysis (may generate internal control gene magnification since mutational site expands Inhibit)；If internal control genetic test (HEX channels) Ct values are larger or without amplification, and mutational site detection (FAM channels) without amplification or There are amplification but Ct values ＞ 38, then can not continue to analyze, need to repeat experiment (degradation may occur due to DNA sample, wherein contain PCR inhibitor is not loaded product).
(4) gene mutation situation is judged in DNA sample to be checked：If certain mutational site detection (FAM channels) has expansion in sample Increasing and Ct value≤38 then judge that sample mutation result is the positive；If Ct values ＞ 38 or without amplification, the sample abrupt junction is judged Fruit is feminine gender.
Pay attention to：Various mutations are may be simultaneously present in same DNA sample.
Fig. 1 is the PCR figures that testing result is negative sample, and Fig. 2 is the PCR figures that testing result is positive sample.
Pass through contrasting detection, it was demonstrated that fluorescence PCR method of the invention and traditional sequencing methods the result is that be consistent, and this The sensitivity of the fluorescence PCR method of invention and high selectivity are in traditional sequencing methods.
Therefore, Fluorescence PCR system of the present invention can detect 23-1728 6 base mutations of missing of C-KIT gene 17s, inspection Survey convenient and efficient, accuracy is high, can meet the requirement that C-KIT gene mutations quickly detect.

Claims (3)

  1. A kind of 1. primer and probe for being used to detect C-KIT gene mutations, which is characterized in that the primer such as SEQ ID No.1- Shown in SEQ ID No.2, the probe is as shown in SEQ ID No.3.
  2. 2. a kind of kit for being used to detect C-KIT gene mutations, which is characterized in that the kit includes SEQ ID No.1- The probe shown in primer and SEQ ID No.3 shown in SEQ ID No.2.
  3. 3. kit as claimed in claim 2, which is characterized in that it further includes reference gene and internal control gene.
CN201510364301.7A 2015-06-29 2015-06-29 A kind of primer, probe and kit for being used to detect C-KIT gene mutations Active CN105112500B (en)

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PCT/CN2016/000351 WO2017000533A1 (en) 2015-06-29 2016-06-27 Primer, probe, and reagent kit for use in detecting mutation in c-kit gene

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Families Citing this family (10)

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Publication number Priority date Publication date Assignee Title
CN107630088A (en) * 2016-07-18 2018-01-26 北京雅康博生物科技有限公司 For detecting primer, probe and the kit of the mutation of C-KIT gene 1648-1686 positions
CN107723360A (en) * 2016-08-11 2018-02-23 嘉兴雅康博医学检验所有限公司 For detecting primer, probe and the kit of 37 1738 mutation of C KIT gene 17s
CN107739757A (en) * 2016-08-11 2018-02-27 嘉兴雅康博医学检验所有限公司 For detecting primer, probe and the kit of 37 1738 mutation of C KIT gene 17s
CN107723361A (en) * 2016-08-11 2018-02-23 北京雅康博生物科技有限公司 For detecting primer, probe and the kit of 1,654 1689 mutation of C KIT genes
CN107630087A (en) * 2016-07-18 2018-01-26 北京雅康博生物科技有限公司 For detecting primer, probe and the kit of 1394 mutation of C-KIT genes
CN107723362A (en) * 2016-08-11 2018-02-23 北京雅康博生物科技有限公司 For detecting primer, probe and the kit of 1,675 1681 mutation of C KIT genes
CN107630089A (en) * 2016-07-18 2018-01-26 嘉兴雅康博医学检验所有限公司 For detecting primer, probe and the kit of the mutation of C-KIT gene 1689-1718 positions
WO2018014519A1 (en) * 2016-07-18 2018-01-25 北京雅康博生物科技有限公司 Primer, probe and kit used for detecting c-kit gene mutation
WO2018014520A1 (en) * 2016-07-18 2018-01-25 嘉兴雅康博医学检验所有限公司 Primers, probes and kit for use in detecting c-kit gene mutations
CN107630090A (en) * 2016-07-18 2018-01-26 嘉兴雅康博医学检验所有限公司 For detecting primer, probe and the kit of 03 1724 mutation of C KIT gene 17s

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