CN102220413B - Method for detecting exon 19 deletion mutation and exon 21 point mutation of epidermal growth factor receptor gene - Google Patents
Method for detecting exon 19 deletion mutation and exon 21 point mutation of epidermal growth factor receptor gene Download PDFInfo
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Abstract
The invention discloses a method for detecting exon 19 deletion mutation and exon 21 point mutation of an epidermal growth factor receptor gene. In method, enzyme digestion enrichment PCR (Polymerase Chain Reaction) reaction and ARMS (amplification refractory mutation system) fluorescence quantitative PCR reaction are carried out in one reaction system to respectively detect E746_A750del deletion mutation and L858R point mutation; and the enzyme digestion mutation enrichment PCR and ARMS fluorescent quantitative PCR are integrated together, the temperature and time are effectively controlled, and one reaction process is carried out to complete the enzyme digestion mutation enrichment and ARMS fluorescent quantitative PCR reaction.
Description
Technical field
The present invention relates to technical field of molecular biology, particularly enzyme is cut enrichment round pcr and ARMS fluorescent quantitative PCR technique.
Background technology
Lung cancer is serious harm people's life and healthy common disease, and the M ﹠ M of at present domestic and international lung cancer is also in continuous rising.The result for the treatment of of lung cancer never significantly improves for many years, and total curative ratio is only 10% left and right, and its reason is on the one hand because its biological characteristics is very complicated, grade malignancy is high and multidrug resistance.Key has also belonged to late period when making a definite diagnosis in the lung cancer more than 80% on the other hand.Late period, the traditional therapy of non-tiny property lung cancer was take chemotherapy as main complex therapy, although constantly there is in recent years novel chemotherapeutics to be found, its curative effect does not significantly improve.A kind of novel target therapeutic agent Iressa (Iressa) is a kind of selectivity EGF-R ELISA (epidermal growth factor receptor, EGFR) tyrosine kinase inhibitor, because its good effect, side effect are little, use in worldwide and come.
Studies show that EGF-R ELISA (EGFR) transgenation is relevant to the susceptibility of antitumor drug Iressa (Iressa) with lung cancer patient.And the front clinical trial phase of lung cancer shows, after the medication combined chemotherapy failure of platiniferous class, Iressa still has that 25%-35%'s is objective efficient, disease control rate is in 50% left and right, median survival interval was about about 7 months, experimenter's doing well,improving and security are good, so the EGFR detection in Gene Mutation has important reference value for the individualized treatment that the clinician carries out lung cancer.Lynch etc. are to effective, invalid and non-tiny cell lung cancer (the non-small cell lung cancer that do not treat with Iressa with Iressa, NSCLC) carried out the detection of EGFR gene, found that 8 examples in 9 routine responders have the sudden change in EGFR gene Tyrosylprotein kinase zone, the sudden change main manifestations of these specific regions is that amino acid whose loss or normal amino acid are replaced; In 7 routine nonresponders, none example has this sudden change, and only has 2 examples that the EGFR transgenation is arranged in the 25 routine patients that do not accept the Iressa treatment, accounts for 8%.The clinical practice demonstration, there is very large individual difference in Iressa to the curative effect of nonsmall-cell lung cancer, only has the individuality of 8-18% to have very good effect.By the mutation analysis to these patient tissues EGFR gene, find most individually undergo mutation in EGFR gene Tyrosylprotein kinase zone.These sudden changes mainly concentrate on exons 1 8-21, and are wherein common with the point mutation L858R of deletion mutantion in 19 exons and 21 exons.Therefore, the sudden change by EGFR gene 18-21 exon detects, and the individualized treatment that carries out lung cancer for prediction targeted drug result for the treatment of and clinician has important reference value.
At present, the DNA sequencing method remains the maximum method of EGFR transgenation use that detects, but need to obtain tumor tissues, separating tumor cell, extraction nucleic acid and check order, required time is long, expense is high, all stricter with technical requirements to drawing materials, therefore be applied to the clinical restriction that still is subjected to a certain extent.There is the laboratory that the detection of EGFR transgenation is provided specially in the U.S., although avoided the restriction that operates and the time that has shortened result, somewhat expensive.At present, people's test kit that domestic clinical unit uses is mainly external import, and is expensive, is unsuitable for domestic use.
Therefore, the purpose of this experiment is exactly take the blood plasma of patients with lung cancer as extracting sample, based on Real-Time Fluorescent Quantitative PCR Technique, provides a kind of easy, quick, accurate and economic lung cancer EGFR detection method of gene mutation for clinical, for the service of lung cancer individualized treatment, for lung cancer patient is benefited.
Summary of the invention
Therefore, one object of the present invention is to provide the method for a kind of detection EGF-R ELISA (EGFR) gene extron 19 deletion mutantion E746_A750del and exon 21 point mutation L858R, utilizes the method can improve the resolving power that detects transgenation.
Another object of the present invention is to provide a kind of test kit that detects epidermal growth factor receptor gene exons 19 deletion mutantion E746_A750del and exon 21 point mutation L858R.
A further object of the present invention is to provide the method for using this test kit to detect epidermal growth factor receptor gene exons 19 deletion mutantion E746_A750del and exon 21 point mutation L858R.
According to one object of the present invention, a kind of method that detects epidermal growth factor receptor gene exons 19 deletion mutantion E746_A750del and exon 21 point mutation L858R is provided, it is characterized in that, carry out enzyme and cut enrichment PCR reaction and ARMS quantitative fluorescent PCR and react and detect respectively exons 19 deletion mutantion E746_A750del and exon 21 point mutation L858R in same reaction system.
In method of the present invention, cut in enrichment PCR reaction at described enzyme, use restriction enzyme Msel and Mscl to carry out enzyme and cut, wherein, described restriction enzyme Msel specific enzymes is cut the TTAA site, and enzyme is cut wild-type exons 19 specifically; Described restriction enzyme Mscl specific enzymes is cut the GCTGGC site, and enzyme is cut wild-type exon 21 specifically, and this endonuclease reaction can reduce wild type gene to the interference of mutated genes.
Particularly, the method for detection epidermal growth factor receptor gene exons 19 deletion mutantion E746_A750del of the present invention and exon 21 point mutation L858R comprises the following steps:
1) preparation detects sample, and the type of preparation sample comprises tissue samples and serum sample, adopts the full DNA extraction test kit of common tissue samples and serum sample can extract target DNA, as Tiangen tissue samples genome DNA extracting reagent kit;
2) carrying out enzyme in same reaction system cuts enrichment PCR reaction and ARMS quantitative fluorescent PCR and reacts and detect respectively exons 19 deletion mutantion E746_A750del and exon 21 point mutation L858R, wherein, cut in enrichment PCR reaction at described enzyme, use restriction enzyme Msel and Mscl to carry out enzyme and cut, and cut at described enzyme the enrichment primer that uses in enrichment PCR reaction and be:
E746_A750del upstream primer: 5 '-CTTCCAAATGAGCTGGCAAGTGC-3 '
E746_A750del downstream primer: 5 '-TGGACCCCCACACAGCAAAGC-3 '
L858R upstream primer: 5 '-CTGCAGAACTTGACCCCTCCCA-3 '
L858R downstream primer: 5 '-GGAAAATGCTGGCTGACCTAAAGC-3 ',
The ARMS primer that uses in described ARMS quantitative fluorescent PCR reaction is:
E746_A750del upstream primer: 5 '-AATTCCCGTCGCTATCAAAAC-3 '
E746_A750del downstream primer: 5 '-ACCCCCACACAGCAAAGC-3 '
L858R upstream primer: 5 '-GTCAAGATCACAGATTTTGCGCG-3 '
L858R downstream primer: 5 '-GGAAAATGCTGGCTGACCTAAAG-3 ',
The Taqman probe sequence that uses in described ARMS quantitative fluorescent PCR reaction is:
E746_A750del probe: 5 '-FAM-CCAACAAGGAAATCCTCGATGTGAGTTTCTG-TAMRA-3 ',
L858R probe: 5 '-FAM-TTCTTTCTCTTCCGCACCCAGCAGT-TAMR-3 '; And
3) interpretation of result is according to Ct (Cycle threshold, threshold cycle number) value result of determination.
In the method for the invention, described step 2) further comprise:
2-1) sample is placed in respectively carries out enzyme and cut the reaction system that enrichment PCR reaction and ARMS quantitative fluorescent PCR react, at 37 ℃ of lower incubation 30min, sample is cut in enrichment ARMS quantitative fluorescent PCR system at enzyme at first carried out endonuclease reaction, specifically wild type gene is carried out enzyme and cut;
2-2) at step 2-1) the basis on, carry out the PCR enrichment, the 400-500bp fragment that can increase has respectively covered the mutational site, the PCR response procedures is: 95 ℃ of 20s, 65 ℃ of 20s, 72 ℃ of 60s, totally 10 circulations; With
2-3) at step 2-2) the basis on, carry out ARMS quantitative fluorescent PCR reaction, mutator gene is carried out Fluorescence Identification, the PCR response procedures is: 95 ℃ of 5s, 60 ℃ of 30s (collection fluorescence), totally 40 circulations.
Wherein, at step 2-2) in, when carrying out the PCR enrichment, described response procedures is hybridized under 65 ℃ the effective renaturation of enrichment primer, and efficient is lower to the ARMS primer; At step 2-3) in, when carrying out the reaction of ARMS quantitative fluorescent PCR, 60 ℃ of extension 30s of described response procedures, only effective to the ARMS primer, the enrichment primer can't form effective amplification with this understanding.
Enzyme is cut enrichment PCR system to method of the present invention and ARMS quantitative fluorescent PCR system is incorporated in a reaction system, control by effective temperature and time, thereby can carry out in a reaction system that enzyme is cut, enrichment PCR and ARMS quantitative fluorescent PCR detect the epidermal growth factor receptor gene sudden change.
In the present invention, the Tm value of described enrichment primer is 65 ℃, and higher 5 ℃ than the Tm value of ARMS primer.
In the present invention, the length of described enrichment primer amplification fragment be ARMS primer amplification fragment length 3-5 doubly.
In the present invention, 3 ' end of described ARMS primer is positioned at purpose mutational site district and is complementary with mutant, and introduces a base mismatch at the 4th of 3 ' end of ARMS primer base place again.
According to another object of the present invention, the invention provides a kind of test kit that detects the epidermal growth factor receptor gene sudden change, it comprises:
(1) enzyme is cut enrichment PCR system, described enzyme is cut enrichment PCR system and is comprised restriction enzyme reaction system and PCR enrichment reaction system, wherein, described restriction enzyme reaction system comprises: restriction enzyme Msel and Mscl, described restriction enzyme Msel specific enzymes is cut the TTAA site, enzyme is cut wild-type exons 19 specifically, described restriction enzyme Mscl specific enzymes is cut the GCTGGC site, enzyme is cut wild-type exon 21 specifically, and this endonuclease reaction can reduce wild type gene to the interference of mutated genes; And the enrichment primer, described enrichment primer can carry out enrichment to the mutational site, and described enrichment primer is:
E746_A750del upstream primer: 5 '-CTTCCAAATGAGCTGGCAAGTGC-3 '
E746_A750del downstream primer: 5 '-TGGACCCCCACACAGCAAAGC-3 '
L858R upstream primer: 5 '-CTGCAGAACTTGACCCCTCCCA-3 '
L858R downstream primer: 5 '-GGAAAATGCTGGCTGACCTAAAGC-3 '.
(2) ARMS quantitative fluorescent PCR system, described ARMS quantitative fluorescent PCR system comprises Taq archaeal dna polymerase, ARMS primer and Taqman probe, described Taq archaeal dna polymerase is without 3 '-5 ' 5 prime excision enzyme activity and extensible 500 bases of per minute, described ARMS primer 3 ' end is positioned at purpose mutational site district, described Taqman probe can carry out fluorescent signal to amplified production and detect, wherein
Described ARMS primer is:
E746_A750del upstream primer: 5 '-AATTCCCGTCGCTATCAAAAC-3 '
E746_A750del downstream primer: 5 '-ACCCCCACACAGCAAAGC-3 '
L858R upstream primer: 5 '-GTCAAGATCACAGATTTTGCGCG-3 '
L858R downstream primer: 5 '-GGAAAATGCTGGCTGACCTAAAG-3 ',
Described Taqman probe sequence is:
E746_A750del probe: 5 '-FAM-CCAACAAGGAAATCCTCGATGTGAGTTTCTG-TAMRA-3 ',
L858R probe: 5 '-FAM-TTCTTTCTCTTCCGCACCCAGCAGT-TAMR-3 '.
Test kit provided by the invention carries out enrichment by above-mentioned PCR enrichment to the mutagenicity gene on the one hand, reduces simultaneously wild type gene reasons for its use signal; By above-mentioned ARMS quantitative fluorescent PCR system, specific amplification is carried out in the mutational site on the other hand, and carry out real-time quantitative by fluorescent signal and detect.
In an embodiment of the invention, described test kit further comprises: Taq archaeal dna polymerase, PCR damping fluid, primer, probe, dNTP, Mg
2+With negative control etc.Wherein, described Taq archaeal dna polymerase is without 3 '-5 ' 5 prime excision enzyme activity and extensible 500 bases of per minute, and described negative control is aseptic deionized water.
According to another object of the present invention, the invention provides a kind of method of using test kit of the present invention to detect the epidermal growth factor receptor gene sudden change, present method has been integrated first enzyme and has been cut enrichment and the reaction of ARMS quantitative fluorescent PCR, comprises the steps:
1) preparation detects sample, and the type of preparation sample comprises tissue samples and serum sample, can adopt the full DNA extraction test kit of common tissue samples and serum sample can extract target DNA, as Tiangen tissue samples genome DNA extracting reagent kit.
2) sample is placed in respectively two and carries out enzyme and cut reaction system that enrichment PCR reaction and ARMS quantitative fluorescent PCR react at 37 ℃ of lower incubation 30min, make sample at first carry out endonuclease reaction, specifically wild type gene is carried out enzyme and cut;
3) in step 2) the basis on, respectively advanced performing PCR enrichment in described two reaction systems, the 400-500bp fragment that can increase has respectively covered the mutational site, the PCR response procedures is: 95 ℃ of 20s, 65 ℃ of 20s, 72 ℃ of 60s, totally 10 circulations; Carry out respectively the reaction of ARMS quantitative fluorescent PCR again, mutator gene is carried out Fluorescence Identification, the PCR response procedures is: 95 ℃ of 5s, 60 ℃ of 30s (collection fluorescence), totally 40 circulations.
In step 3) in, when carrying out the PCR enrichment, described response procedures is hybridized under 65 ℃ the effective renaturation of enrichment primer, and efficient is lower to the ARMS primer; When carrying out the reaction of ARMS quantitative fluorescent PCR, 60 ℃ of extension 30s of described response procedures, only effective to the ARMS primer, the enrichment primer can't form effective amplification with this understanding.
4) interpretation of result is according to Ct (Cycle threshold, threshold cycle number) value result of determination.
Real-Time Fluorescent Quantitative PCR Technique in the present invention refers to add fluorophor in the PCR reaction system, utilizes the whole PCR process of fluorescent signal accumulation Real-Time Monitoring, by typical curve, unknown template is carried out at last the method for quantitative analysis.
Amplification refractory mutation system in the present invention (amplification refractory mutation system, ARMS) set up in 1989, it is the development that round pcr is used, also claim allele specific amplification method (Allele-specific amplification, ASA), allelotrope characteristic PCR (allele-specific PCR, ASPCR) etc., it uses for reference multiplex PCR principle, can detect simultaneously two or more allelic mutations sites in same system.The method is by two 5 ' end primers of design, one complementary with normal DNA, and one complementary with mutant DNA, suddenlys change for homozygosity, add respectively these two kinds of primers and 3 ' end primer to carry out two parallel PCR, only have extensible with the primer ability of the complete complementation of mutant DNA and obtain pcr amplification product.If mispairing is positioned at 3 ' end of primer can causes PCR not extend.
The present invention is incorporated in a reaction system by enzyme being cut enrichment round pcr and ARMS fluorescent quantitative PCR technique, can realize enrichment and evaluation to transgenation having reduced operation steps by a step, has improved detection sensitivity and specificity.
Description of drawings
Fig. 1 is for using method of the present invention to detect the positive findings of epidermal growth factor receptor gene exons 19 deletion mutantion E746_A750del.
Fig. 2 is for using method of the present invention to detect the positive findings of epidermal growth factor receptor gene exon 21 point mutation L858R.
Embodiment
The invention will be further described below in conjunction with the drawings and specific embodiments, but the present invention is not limited to following examples.
In following embodiment, if no special instructions, be ordinary method.
One, material
Restriction enzyme Msel and Mscl be available from Britain NEB company, and (this Mix has comprised PCR damping fluid, dNTP, Mg to 2 * Universe Taqman Mix available from American AB I company
2+, provide by supplier), the primer and probe are given birth to work by Shanghai and are synthesized, and Tiangen tissue samples genome DNA extracting reagent kit is followed bio tech ltd available from sky, Beijing.Instrument is the American AB I ABI7300 of company quantitative real time PCR Instrument.
Two, primer and probe sequence design
1, enrichment design of primers
Design respectively the primer of the one section 400-500bp that can increase, correlation parameter is: 65.0 ℃-68.0 ℃ of Tm values, GC value 40.0%-65.0%, primer size 23 ± 3bp.Designed enrichment primer sequence is as follows:
E746_A750del upstream primer: 5 '-CTTCCAAATGAGCTGGCAAGTGC-3 ' (SEQID No.1)
E746_A750del downstream primer: 5 '-TGGACCCCCACACAGCAAAGC-3 ' (SEQID No.2)
L858R upstream primer: 5 '-CTGCAGAACTTGACCCCTCCCA-3 ' (SEQ ID No.3)
L858R downstream primer: 5 '-GGAAAATGCTGGCTGACCTAAAGC-3 ' (SEQ IDNo.4)
2, ARMS design of primers
Design respectively the primer of the one section 80-120bp that can increase, correlation parameter is: 55.0 ℃-60.0 ℃ of Tm values, GC value 40.0%-60.0%, primer size 20 ± 3bp.3 ' end of ARMS primer is positioned at the mutational site district, and is complementary with mutant, be to improve specificity, and the place introduces a base mismatch again in the 4th of 3 ' end base, and designed ARMS primer sequence is as follows:
E746_A750del upstream primer: 5 '-AATTCCCGTCGCTATCAAAAC-3 ' (SEQ IDNo.5)
E746_A750del downstream primer: 5 '-ACCCCCACACAGCAAAGC-3 ' (SEQ IDNo.6)
L858R upstream primer: 5 '-GTCAAGATCACAGATTTTGCGCG-3 ' (SEQ IDNo.7)
L858R downstream primer: 5 '-GGAAAATGCTGGCTGACCTAAAG-3 ' (SEQ IDNo.8)
3, probe design
Designing probe in ARMS primer amplification fragment respectively, correlation parameter is: 68.0 ℃-70.0 ℃ of Tm values, GC value 40.0%-70.0% carries out 5 ' end FAM mark to probe, 3 ' end TAMRA mark, its sequence is as follows:
E746_A750del probe: 5 '-CCAACAAGGAAATCCTCGATGTGAGTTTCTG-3 ' (SEQ ID No.9)
L858R probe: 5 '-TTCTTTCTCTTCCGCACCCAGCAGT-3 ' (SEQ IDNo.10).
The present method of utilizing embodiment 2 detects E746_A750del and L858R sudden change
The detection method of sudden change enrichment ARMS quantitative fluorescent PCR of the present invention has been integrated enzyme and has been cut enrichment round pcr and ARMS fluorescent quantitative PCR technique, namely in a reaction system to sample carry out respectively that enzyme is cut, PCR enrichment and ARMS quantitative fluorescent PCR identify.Detailed process is:
1, sample preparation: adopt Tiangen tissue samples genome DNA extracting reagent kit to extract sample genomic dna.
2, detection method: at first with sample and negative control respectively in 25 μ l reaction systems under 37 ℃ temperature bathe 30min, use restriction endonuclease Msel (NEB, England) or Mscl (NEB, England) respectively EGFR wild- type exons 19 or 21 is carried out enzyme and cut, to reduce wild-type DNA to the interference of mutating alkali yl.Then carry out the PCR reaction, move first circulation mutating alkali yl is carried out enrichment, under 65 ℃ of annealing temperatures, the ARMS primer can not effectively be worked, and therefore only has the enrichment primer to extend; Move second circulation time, extend 30s at 60 ℃, because selected ABI Taq enzyme per minute extends 500 bases, in 30s, the enrichment primer can not effectively extend, and does not become circulation, therefore only carries out the ARMS fluorescent reaction.The component that 25 μ l reaction systems of detection E746_A750del and L858R sudden change comprise is distinguished as shown in Table 1 and Table 2:
Table 1
Table 2
The PCR response procedures is: 37 ℃ of temperature are bathed 30min; 95 ℃ of 10min; First circulation: 95 ℃ of 20s, 65 ℃ of 20,72 ℃ of 60s, totally 10 circulations; Second circulation: 95 ℃ of 5s, 60 ℃ of 30s (collection fluorescence), totally 40 circulations.
Negative control in the method is aseptic deionized water.
3, interpretation of result: Fig. 1 is for detecting the positive findings of epidermal growth factor receptor gene exons 19 deletion mutantion E746_A750del.Fig. 2 is for detecting the positive findings of epidermal growth factor receptor gene exon 21 point mutation L858R.Wherein, the Ct value is judged as positive findings less than 36, and the Ct value must detect between 36-38 again, and the Ct value is judged as feminine gender greater than 38 or institute's complete genome DNA concentration of carrying is too low, exceeds sensing range.
1, materials and methods
Identify that from Concord Hospital and Chinese pharmaceutical biological product institute collects the tumor tissues sample of 73 parts of lung cancer altogether, adopt Tiangen tissue samples genome DNA extracting reagent kit extraction complete genome DNA.
Respectively get the 2 μ l enrichment ARMS fluorescence quantitative PCR detection of suddenling change, do negative control with aseptic deionized water, specific implementation method is seen embodiment 2.
2, experimental result
In 73 parts of tissue samples, detect altogether 12 parts of E746_A750del deletion mutantions, 11 parts of L858R sudden changes.Detection method is judged identical with embodiment 2.
Claims (1)
1. a test kit that detects epidermal growth factor receptor gene exons 19 deletion mutantion E746_A750del and exon 21 point mutation L858R, is characterized in that, this test kit comprises:
(1) enzyme is cut enrichment PCR system, and described enzyme is cut enrichment PCR system and comprised restriction enzyme Msel and Mscl; And the enrichment primer, described enrichment primer is:
E746_A750del upstream primer: 5 '-CTTCCAAATGAGCTGGCAAGTGC-3 '
E746_A750del downstream primer: 5 '-TGGACCCCCACACAGCAAAGC-3 '
L858R upstream primer: 5 '-CTGCAGAACTTGACCCCTCCCA-3 '
L858R downstream primer: 5 '-GGAAAATGCTGGCTGACCTAAAGC-3 ';
(2) ARMS quantitative fluorescent PCR system, described ARMS quantitative fluorescent PCR system comprises Taqman probe and ARMS primer, wherein said ARMS primer is:
E746_A750del upstream primer: 5 '-AATTCCCGTCGCTATCAAAAC-3 '
E746_A750del downstream primer: 5 '-ACCCCCACACAGCAAAGC-3 '
L858R upstream primer: 5 '-GTCAAGATCACAGATTTTGCGCG-3 '
L858R downstream primer: 5 '-GGAAAATGCTGGCTGACCTAAAG-3 ',
Described Taqman probe sequence is:
E746_A750del probe: 5 '-FAM-CCAACAAGGAAATCCTCGATGTGAGTTTCTG-TAMRA-3 ',
L858R probe: 5 '-FAM-TTCTTTCTCTTCCGCACCCAGCAGT-TAMRA-3 '.
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