CN101041850A - T790M mutation quick-detection method and reagent case for human epidermal growth factor acceptor(EGFR) gene extron 20 - Google Patents
T790M mutation quick-detection method and reagent case for human epidermal growth factor acceptor(EGFR) gene extron 20 Download PDFInfo
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Abstract
The invention discloses a T790M abrupt change fast testing method of human cuticle growth factor acceptor (EGFR) gene extron 20 in molecular biology domain, which comprises the following steps: designing one primer according to DNA sequence of human colorant layer 7p12 EGFR gene extron; adopting specificity of polyose chain manner reaction (PCR) technology; augmenting a flight of DNA sequence with T790M(2369 C>T) site point of discontinuity; utilizing a molecular fluorescence probe and augmentation product to cross; real-time-checking relevant fluorescence intensity change of characteristic of reacted pipe through fluorescence PCR device; or direct-testing specificity fluorescence strength of reacted pipe through fluorescence spectrophotometer; testing T790M abrupt change of specimen. This invention is simple and fast, which also provides agent box for clinic.
Description
Technical field the invention belongs to biology field.
Background technology in June, 2004, American scholar Lynch and Paez etc. at first report, the sudden change of nonsmall-cell lung cancer mesocuticle growth factor receptors (EGFR) Tyrosylprotein kinase coding region gene is the prerequisite that targeted drug proves effective, confirmed EGFR coding region gene mutant tumour, EGFR tyrosine kinase inhibitor gefitinib's (claiming Iressa or ZD1839 again) is efficient up to more than 80%, and invalid substantially to the wild-type tumour said medicine of not having sudden change.Moreover, treat effective patient through gefitinib, (drug failure then during the sudden change of 2369 C＞T) needs effective inhibitor that in time more renew or uses other methods of treatment when T790M appears in the EGFR gene coding region.Therefore,, can from the advanced lung cancer patient, filter out the suitableeest treatment target and carry out individualized treatment, thereby significantly improve curative effect of medication, economize on resources by detecting the EGFR genic mutation type.
Advantage of the present invention
Round pcr with himself cleverly principle distinguished characteristics are arranged, high specific, hypersensitivity and simple and efficient one of its technology that becomes the gene diagnosis first-selection that makes.
(1) high specific, pcr amplification strictly observe the semiconservative replication of basepairing rule.Semiconservative replication is one of the strictest in the world copy mode, and its new synthetic subchain and template form complete complementary mirror-image structure, thereby has fully guaranteed the accuracy of duplicating.In addition, because base complementrity principle, have only when primer and the complete complementation of goal gene, primer in the reaction system could produce renaturation with template, the extension of primer is just carried out, therefore the complementation of primer and template is the fundamental prerequisite that duplicates, and this has stipulated the high specific of PCR reaction from another point of view.In organic sphere, certain gene always has its most conservative, distinctive constant gene segment C of tool, and it is some biology, or function somatotypes such as some type, hypotype are peculiar.If can correctly select the goal gene of this zone as amplification, the specificity of joint probe hybridization just can ensure the high degree of specificity that PCR detects fully.
(2) hypersensitivity, template DNA increases sharply with exponential in the PCR reaction, amplified reaction enters in earlier stage with exponential and increases sharply, the amplified reaction later stage enters the flat reaction phase, be target sequence can be increased more than 1,000,000 times in 1-2 hour generally, the target compound (fg DNA) of trace can be detected through 30 circulations.Some trace detection methods that past adopts be respectively ng level and pg level as enzyme-linked immunosorbent assay (ELISA) and its sensitivity of radioimmunoassay (RIA), and PCR can reach the fg level, can detect the existence of the single copy gene of pathogenic agent in theory.
(3) simple and efficient, the use of Taq enzyme can be finished round pcr in automatization, and continuous mutually appearance of various High Efficiency PC R instrument can be finished the PCR operation smoothly in the laboratory of grass-roots unit.In the practical application of PCR, many technology are improved, and the amplified reaction volume reduces, and multiple composition is pre-mixed and reduces the application of sample step, and these simplify the expanding effect that step scarcely influences PCR.The single part PCR reagent of single tube particularly, make operator's energy of saving time, and be difficult for again polluting, that has fully satisfied clinical quick diagnosis requires round pcr low to sample requirement, needn't strict purifying template DNA, nearly all clinical sample can both be used for pcr amplification.
(4) economical and practical, traditional SNP or point mutation detecting method such as PCR-RFLP, PCR-SSCP, PCR-SSO etc., relate to the aftertreatment such as cut, develop the color and take pictures of electrophoresis, enzyme mostly, loaded down with trivial details during operational cost, specificity and susceptibility are relatively poor, can not automatization and high throughput testing, also cause crossed contamination easily, order-checking and chip technology costliness then consuming time.The present invention combines the characteristic of PCR and molecular probe, the accurately T790M of the rapid detection EGFR gene extron 20 (sudden change of 2369 C＞T), especially the real-time detection technique of PCR can make amplified reaction and molecular hybridization carry out in the system of a sealing, need not the PCR post-processing step, avoided pollution, and can differentiate single base difference, highly sensitive, easy and simple to handle quick, be suitable for the detection of sample in enormous quantities, whole detection can be finished at 2 hours.China's malignant tumour is first reason of human mortality, and wherein the sickness rate with lung cancer, cancer of the stomach and the esophageal carcinoma is the highest, accounts for more than 60% of the dead sum of malignant tumour.Current each large, medium and small medical institutions have all disposed the fluorescent PCR instrument basically, and the present invention has extremely wide application prospect.
Summary of the invention the invention provides the T790M mutation quick-detection method and the test kit of a kind of human epidermal growth factor acceptor (EGFR) gene extron 20, what its feature adopted pcr amplification human chromosomal 7p12 EGFR gene extron 20 comprises the T790M (gene order in mutational site of 2369 C＞T), use the hybridization of molecular fluorescence probe and amplified production, use the fluorescent PCR instrument and measure the characteristic fluorescence signal intensity variation of reaction solution in the PCR process in real time, or use the specific fluorescence intensity that the fluorescence spectrophotometry instrument is measured reaction solution behind the PCR, carry out the result and judge.
CDNA sequence selection primer according to Human epidermal growth factor receptor (EGFR) gene Seq ID NO:NM_005228 is: the EGFR gene extron 20 that can increase comprise T790M (a pair of primer of the gene order in mutational site of 2369 C＞T); The selection probe is: two oligonucleotide probes of design and target complement sequence in two primer amplification fragments, and 5 ' end is modified with FAM or HEX respectively, and 3 ' holds with amido modified and mark DABCYL.Its sequence is:
Upstream primer F:5 ' CCGCCTGCTGGGCATCTG 3 '
Downstream primer R:5 ' GCGATCTGCACACACAGTTGAG 3 '.
Wild-type probe sequence: Probe-wt:FAM-5 '-GCGACATCACGCAGCTCATGCCCTGTCGC-3 '-DABCYL
Mutant probe sequence: Probe-mu:HEX-5 '-GCGACATCATGCAGCTCATGCCCTGTCGC-3 '-DABCYL
(2369 C＞T) test kit of sudden change comprises: reaction mixture to detect T790M; The mutant contrast; The wild-type contrast; Sample extracting solution; Working instructions.Test kit mutant contrast is T790M (the mutant DNA sequence of 2369 C＞T); Wild-type contrast is T790M (the wild-type dna sequence dna of 2369 C＞T).The test kit reaction mixture is: 1 * PCR damping fluid (0.1%TritonX-100; 2.0～6.0mmol/L MgCl
28mmol/L (NH
4)
2SO
450mmol/L KCl; 10mmol/L Tris-HCl (pH8.0)); Include each 0.3 μ mol/L of two primers, Probe-wt0.2 μ mol/L, Probe-mu0.3 μ mol/L; Each 250 μ mol/L of dNTP, HotTaq enzyme 1.5U, reaction cumulative volume 50 μ l.
The present invention protects designed primer and probe sequence to be applied to T790M (2369 C＞T) sudden change of hybridization hybrid chip detection human chromosomal 7p12EGFR gene extron 20.
Description of drawings Fig. 1 is the pcr amplification conditional parameter; The fluorescent signal growth curve form that Fig. 2 may occur when detecting for the fluorescent PCR instrument.
Embodiment test kit of the present invention is composed as follows:
Reaction mixture: 1 * PCR damping fluid (0.1%TritonX-100; 2.0～6.0mmol/L MgCl
28mmol/L (NH
4)
2SO
450mmol/L KCl; 10mmol/LTris-HCl (pH8.0)); Include each 0.3 μ mol/L of two primers, Probe-wt0.2 μ mol/L, Probe-mu0.3 μ mol/L; Each 250 μ mol/L of dNTP, HotTaq enzyme 1.0U, reaction cumulative volume 50 μ l.Positive control is T790M (the mutant DNA sequence of 2369 C＞T); Negative control is its wild-type dna sequence dna.
Sample extracting solution: (1). dimethylbenzene; (2). dehydrated alcohol; (3) .1.5% trolamine lauric acid vitriol (TLS); (4). chloroform: primary isoamyl alcohol (24: 1); (6) .TE (pH8.0).
Wild-type probe sequence: Probe-wt:FAM-5 '-GCGACATCACGCAGCTCATGCCCTGTCGC-3 '-DABCYL
Mutant probe sequence: Probe-mu:HEX-5 '-GCGACATCATGCAGCTCATGCCCTGTCGC-3 '-DABCYL
Entrust Shanghai Shen You Bioisystech Co., Ltd synthetic.
The PCR primer: company limited is synthetic by the precious biotechnology in Dalian.
Upstream primer F:5 ' CCGCCTGCTGGGCATCTG 3 '
Downstream primer R:5 ' GCGATCTGCACACACAGTTGAG 3 '.
HotTaq enzyme: available from the precious biotechnology in Dalian company limited.
DNTPs (dATP, dTTP, dCTP, dGTP): give birth to worker bio-engineering corporation available from Shanghai.
PCR thin-walled reaction tubes: available from the precious biotechnology in Dalian company limited, 200 μ l.
Detect step:
(1) tissue DNA extracts
Section is taken off cured: get the eppendorf pipe that the thick tissue slice of 3～5um about 300mg is put into 1.5ml, add 1ml dimethylbenzene, the vortex vibration centrifugal 5 minutes, is abandoned supernatant; Repeat twice of dimethylbenzene extracting again.With 1ml absolute ethanol washing precipitation sheet, vortex vibration, centrifugal 5 minutes, abandon supernatant, repeated washing is once.Thorough drying.
Fully shred or grind an amount of tissue of slurry (about 300mg), paraffin embedding or section must routine be taken off cured, move into the eppendorf pipe of 5ml, add physiological saline 200ul, 1.5% trolamine lauryl sulfate (TLS) 200ul, 0 ℃ 1 hour, add the equal-volume chloroform: primary isoamyl alcohol (24: 1v/v), shake well 10min, 3000r/min 10min, sucking-off upper strata water is gone into new pipe, adds the equal-volume chloroform: primary isoamyl alcohol (24: 1 v/v), shake well 10min, 3000r/min 10min, sucking-off upper strata water adds the cold ethanol of equal-volume, the centrifugal 10000r/min 1min of mixing, abandon supernatant, get precipitation, be dissolved in TE (pH8.0) 20～30ul and be high purity DNA, 4 ℃ stand-by.
(2) pcr amplification reaction
(1) pcr amplification reaction mixed solution
Get the above-mentioned DNA extraction liquid of 2ul as template, add 1 * PCR damping fluid (0.1%TritonX-100; 2.0～6.0mmol/LMgCl
28mmol/L (NH
4)
2SO
450mmol/L KCl; 10mmol/LTris-HCl (pH8.0)); Include each 0.3 μ mol/L of two primers, Probe-wt0.2 μ mol/L, Probe-mu0.3 μ mol/L; Each 250 μ mol/L of dNTP, HotTaq enzyme 1.0U, reaction cumulative volume 50 μ l.
(2) pcr amplification condition (as shown in Figure 1)
(3) result judges
(1) the real-time detection of fluorescent PCR instrument
Be set in the characteristic fluorescence signal that 58 ℃ of annealing the time detect FAM and HEX fluorescein simultaneously in the PCR process and increase, if only have the signal of FAM fluorescein to be the characteristic exponential growth and pass thresholding then illustrate that this sample is a homozygous wildtype; If only have the signal of HEX fluorescein to be the characteristic exponential growth and pass thresholding then illustrate that this sample is T790M (the homozygous mutation type of 2369 C＞T); If the signal of FAM and HEX fluorescein all is the characteristic exponential growth and passes thresholding then illustrate that this sample is T790M (the heterozygous mutant type of 2369 C＞T).(as shown in Figure 2)
(2) spectrophotofluorometer detects
If after not having the PCR reaction to finish, spectrophotofluorometer is in the fluorescence intensity of 58 ℃ of difference detection reaction pipe FAM and HEX fluorescein, corresponding fluorescence intensity with feminine gender and positive control is contrast respectively, and fluorescence intensity is higher than 4 times of standard deviations of corresponding background fluorescence intensity as meaningful judgement.The final sudden change sex of judging template DNA.
Claims (6)
1. the T790M mutation quick-detection method of a human epidermal growth factor acceptor (EGFR) gene extron 20, what its feature adopted pcr amplification human chromosomal 7p12 EGFR gene extron 20 comprises the T790M (gene order in mutational site of 2369 C＞T), use the hybridization of molecular fluorescence probe and amplified production, use the fluorescent PCR instrument and measure the characteristic fluorescence signal intensity variation of reaction solution in the PCR process in real time, or use the specific fluorescence intensity that the fluorescence spectrophotometry instrument is measured reaction solution behind the PCR, carry out the result and judge.
2. according to the T790M mutation quick-detection method of a kind of human epidermal growth factor acceptor (EGFR) gene extron 20 of claim 1, it is characterized in that selecting primer to be: the EGFR gene extron 20 that can increase comprise T790M (a pair of primer of the gene order in mutational site of 2369 C＞T):
Upstream primer F:5 ' CCGCCTGCTGGGCATCTG 3 '
Downstream primer R:5 ' GCGATCTGCACACACAGTTGAG 3 '.
3. according to the T790M mutation quick-detection method of a kind of human epidermal growth factor acceptor (EGFR) gene extron 20 of claim 1, it is characterized in that it detects T790M (the molecular fluorescence probe of sudden change of 2369 C＞T): two oligonucleotide probes of design and target complement sequence in two primer amplification fragments, 5 ' end is modified with FAM or HEX respectively, and 3 ' holds with amido modified and mark DABCYL.Its sequence is:
Wild-type probe sequence: Probe-wt:FAM-5 '-GCGACATCACGCAGCTCATGCCCTGTCGC-3 '-DABCYL
Mutant probe sequence: Probe-mu:HEX-5 '-GCGACATCATGCAGCTCATGCCCTGTCGC-3 '-DABCYL.
4. according to the T790M mutation quick-detection method of a kind of human epidermal growth factor acceptor (EGFR) gene extron 20 of claim 1, (2369 C＞T) test kit of sudden change comprises: reaction mixture to it is characterized in that detecting T790M by fluorescent probe PCR; The mutant contrast; The wild-type contrast; Sample extracting solution; Working instructions.
5. according to the T790M mutation quick-detection method of a kind of human epidermal growth factor acceptor (EGFR) gene extron 20 of claim 1, (2369 C＞T) the test kit reaction mixture of sudden change is: 1 * PCR damping fluid (0.1%TritonX-100 to it is characterized in that detecting T790M; 2.0～6.0mmol/L MgCl
28mmol/L (NH
4)
2SO
450mmol/L KCl; 10mmol/L Tris-HCl (pH8.0)); Include each 0.3 μ mol/L of two primers, Probe-wt0.2 μ mol/L, Probe-mu0.3 μ mol/L; Each 250 μ mol/L of dNTP, HotTaq enzyme 1.5U, reaction cumulative volume 50 μ l.
6. according to the T790M mutation quick-detection method of a kind of human epidermal growth factor acceptor (EGFR) gene extron 20 of claim 1, it is characterized in that: designed probe sequence is applied to T790M (2369 C＞T) sudden change that hybridization hybrid chip detects human chromosomal 7p12EGFR gene extron 20.
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| CN101463390B (en) * | 2008-12-31 | 2011-09-14 | 广州益善生物技术有限公司 | EGFR gene extron 20 mutational detecting probe, liquid phase chip and detecting method thereof |
| CN102234683A (en) * | 2010-04-23 | 2011-11-09 | 广州益善生物技术有限公司 | Liquid phase chip for detecting EGFR (epidermal growth factor receptor) gene mutation |
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| CN101613698B (en) * | 2009-04-13 | 2011-06-15 | 厦门艾德生物医药科技有限公司 | Primer design method for amplifying low-content gene mutation DNA and application thereof |
| CN102234683A (en) * | 2010-04-23 | 2011-11-09 | 广州益善生物技术有限公司 | Liquid phase chip for detecting EGFR (epidermal growth factor receptor) gene mutation |
| CN102234683B (en) * | 2010-04-23 | 2013-07-17 | 广州益善生物技术有限公司 | Liquid phase chip for detecting EGFR (epidermal growth factor receptor) gene mutation |
| CN103282515A (en) * | 2010-12-22 | 2013-09-04 | 霍夫曼-拉罗奇有限公司 | Methods and compositions for detecting mutation in the human epidermal growth factor receptor gene |
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| CN103255201A (en) * | 2012-02-16 | 2013-08-21 | 北京宏微特斯生物科技有限公司 | Method of detecting gene mutation based on Blocker primers and ARMS primers, and kit |
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| CN107419018B (en) * | 2012-02-16 | 2020-09-04 | 江苏宏微特斯医药科技有限公司 | Method and kit for detecting gene mutation based on Blocker primer and ARMS primer |
| CN103045746A (en) * | 2012-12-31 | 2013-04-17 | 上海市胸科医院 | Amplification primer, detection probe and liquid phase chip for EGFR gene mutation detection |
| CN103923974A (en) * | 2014-01-27 | 2014-07-16 | 上海涌泰生物医药科技有限公司 | Kit for detecting T790M site mutation of EGFR gene exon 20, and method thereof |
| CN103923974B (en) * | 2014-01-27 | 2016-02-03 | 上海涌泰生物医药科技有限公司 | A kind of test kit and method detecting EGFR gene extron 20 T790M point mutation |
| CN104531874A (en) * | 2014-12-29 | 2015-04-22 | 深圳华因康基因科技有限公司 | EGFR gene mutation detecting method and kit |
| CN104498616A (en) * | 2015-01-09 | 2015-04-08 | 湖南圣湘生物科技有限公司 | Human EGFR gene mutation fluorescent PCR detection kit |
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| CN111876472B (en) * | 2020-06-17 | 2023-12-01 | 江门市灿明生物科技有限公司 | Method for detecting trace nucleic acid in multiple mixed nucleic acids |
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