CN101608240A - Be used to detect primer, probe and the using method thereof of human EGFR gene sudden change - Google Patents
Be used to detect primer, probe and the using method thereof of human EGFR gene sudden change Download PDFInfo
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Abstract
The invention discloses the primer, probe and the using method that are used to detect the human EGFR gene sudden change, relate to the detection of transgenation.Method of the present invention comprises that (1) provides primer and probe; (2) testing sample is handled and template extraction; (3) reaction system of preparation fluorescent PCR amplification mutator gene sequence; (4) primer and the probe amplification mutator gene target sequence to be measured of usefulness step (1); (5) utilize the hybridization of dicyclo probe and amplified production, the FAM of detection reaction system and HEX or the consequent judging criterion of ROX fluorescence intensity.Primer of the present invention, probe and method: can detect 29 deletion mutantions of EGFR gene simultaneously; Highly sensitive; High specificity; The selectivity ability is strong; Detection speed is fast, and whole testing process only needs 90 minutes.
Description
Technical field
The present invention relates to detection in Gene Mutation, particularly detect primer, probe and the method for 29 kinds of deletion mutantions of human epidermal growth factor acceptor EGFR gene.
Background technology
(epidermal growth factor receptor EGFR) is present in the cell that leaf is originated between all normal epithelial and part to human epidermal growth factor acceptor, and the growth of pair cell, the adjusting of differentiation play an important role.EGFR can activate Tyrosylprotein kinase, thereby opens the downstream signal path.Therefore, EGFR becomes an especially important target spot of nonsmall-cell lung cancer targeted therapy research of tumour.At present, the EGFR targeted therapy mainly comprises small molecules tyrosine kinase inhibitor and two parts of monoclonal antibody clinically, and as the Gefitinib (Iressa) of Astrazeneca AB, evil sieve of Roche Holding Ag is for Buddhist nun (Te Luokai).
Discover the coding region of some lung cancer patient EGFR genes in recent years in a large number, mainly be on exon 18-21, can undergo mutation, and these sudden changes are relevant for the clinical efficacy of medicines such as Buddhist nun with Gefitinib and evil sieve, promptly relevant with drug responsiveness, reason may be because these sudden changes have changed the structure of ATP-binding domain in the EGFR born of the same parents, has improved the binding ability of EGFR to Gefitinib.At present, though can not have been found that any more 30 kinds of sudden changes, mainly be the point mutation of L858R on deletion mutantion on the exons 19 and the exon 21; The different disappearances of nearly more than 20 kinds of 747-750 amino acids account for 45% of sudden change on the exons 19, and are wherein common with 2235_2249del15 and 2236_2250del15; The alternative 40-45% that accounts for sudden change of 858 amino acids on the exon 21; Exons 18 point mutation (G719S or G719C) account for 5% of sudden change; Insertion sudden change on the extron 20 accounts for about 1% of sudden change.The G719X sudden change of exons 18 accounts for about 5% of sudden change.But the mutation rate of EGFR is not high in patients with lung cancer, and the mutation rate of EGFR gene only has 2% in U.S.'s lung cancer patient, and Japan is 40%.The mutation rate of EGFR gene is 30% among the patient of Chinese women nonsmall-cell lung cancer, if this part patient all can use Gefitinib or dislike sieve and will obviously improve prognosis for Buddhist nun's treatment.
Though these targeted drugs cost an arm and a leg to some crowd's curative effect very much,, not only delay the waste that the state of an illness also causes huge expenses for medicine if patient does not carry the EGFR sudden change and takes this type of medicine.Therefore, whether carrying the EGFR transgenation medication fore worker inspection survey patient and seem particularly important, also is the developing direction of following tumour individualized treatment.So how detecting these EGFR transgenations relevant with drug responsiveness rapidly and accurately becomes people's problem demanding prompt solution.
Foundation is different from the general genetic mutation of detection to tumour patient EGFR detection method of gene mutation, even if general heredity sudden change heterozygosity sudden change (heterogeneity) and wild ratio are 1/1 forever, just can be by general probe technique to suddenling change and wildly well distinguishing.Yet these EGFR sudden changes all are somatic mutations, extract in the template to have a large amount of wild-type DNA, and the sudden change proportion is uncertain, and directly from the sample of excision, the mutant proportion height may reach more than 20%, and the content that perhaps suddenlys change has only 0.1%.Though can catch tumour cell in the sample by micro laser, improve the ratio of tumour cell in the sample.Even if tumour cell but, tumour cell that neither 100% all has this type of sudden change, and the cell that is in different tumor tissues position is perhaps not quite alike.Perhaps can be lower if come the sample of autoblood to suddenly change content.The difficult point of rare mutation detection that Here it is: one side will have high-sensitive detectivity---and units even single sudden change copy must guarantee high specificity in addition---face the interference of thousands of times up to ten thousand times wild-type templates, false positive do not occur.
The present method that detects the EGFR gene rare mutation has multiple, and detectivity is 1%～20%.Mainly contain two big classes, a class is non-PCR in real time, mainly contains order-checking and DHPLC, and detectivity only is 20% and 5%, and needs the PCR aftertreatment, complex steps, and length consuming time is polluted easily; Another kind of is PCR in real time, though all be based on the technology platform of PCR in real time, in order to improve the selective power of detection method, the used strategy of different researchers is not quite similar.Mainly contain and rely on dna sequence dna self identification specificity fully and rely on enzyme identification specificity two big class strategies, TaqMan probe, the analysis of high resolution melting curve, competitive inhibition real-time PCR method are arranged, between the detectivity 5%～20%, DxS scorpion primer method detectivity can reach 1%.
In recent years, discover in patient's peripheral blood, to detect the mutant DNA that cancer cells is overflowed, become the biomarker of good early diagnosis of tumor, treatment prognosis.Kimura etc. (2006) report can detect the EGFR sudden change in circulation blood serum.Think and detect mutant DNA in these peripheral bloods, can know the molecules feature in targeted drug target site, thereby judge which medicine is the most effective to patient.But, the mutant DNA of cancer cells is the very rare mutator gene that is present under a large amount of wild type gene backgrounds in the peripheral blood, it exists ratio to be generally less than 0.2%, even if also mixed a large amount of normal cells in the cancerous lung tissue of excision, and not all tumour cell all has sudden change.Can aforesaid method can not satisfy increasing noninvasive sudden change and detect needs, therefore accurately do not have this class rare mutation of wound ground detection existing detection technique has been proposed challenge.
Summary of the invention
Limited in order to overcome the prior art detectivity, a kind of fluorescence PCR method that detects 29 kinds of sudden changes of human epidermal growth factor acceptor EGFR gene now is provided.
For solving the problems of the technologies described above, technical scheme of the present invention is, a kind of fluorescence PCR method that detects 29 kinds of sudden changes of human epidermal growth factor acceptor EGFR gene, and it may further comprise the steps:
(1) according to the wild type gene sequence of human epidermal growth factor acceptor (EGFR) gene 18,19,20,21 exons and and corresponding mutator gene sequence, design many to high specific degenerated primer and a plurality of specificity dicyclo probe.
(2) reaction system of preparation fluorescent PCR amplification mutator gene sequence.
(3) with each the specific specificity degenerated primer of step (1) and the specificity dicyclo probe 29 kinds of deletion mutantion gene orders to be measured that increase simultaneously.
(4) utilize the hybridization of dicyclo probe and amplified production, the FAM of detection reaction system and HEX or ROX fluorescence intensity show sample DNA amount in allowed band when reaching setting threshold (Ct＞18) with HEX (or ROX) signal, and the FAM signal results is credible; The standard that needed cycle index Ct value is judged as yin and yang attribute when reaching preset threshold with FAM, the Ct value is 0 or 35: feminine gender; Ct is less than 32: the positive.The reaction system of fluorescent PCR is:
1 * PCR damping fluid
MgCl
2 5.0～10.0mmol
Each 0.8～1.5mmol of dNTP
Each is to primer 0.1 ~ 1.0 μ mol
Each bar probe 0.5 ~ 1.0 μ mol
Taq enzyme 2.0～3.0U
Template 4.0～6.0 μ l
The reaction conditions of described fluorescent PCR is 95 ℃ of pre-sex change 5 minutes, 94 ℃ of sex change of 15 circulations 15 seconds, annealed 40 seconds for 66 ℃, 72 ℃ were extended 10 seconds, 94 ℃ of sex change of 35 circulations 15 seconds were annealed 40 seconds for 56 ℃, and 72 ℃ were extended 10 seconds, 35 circulations, back 35 circulate in detection FAM and HEX or ROX fluorescent signal when annealing.
Described deletion mutantion is 29 kinds of common mutations of patients with lung cancer EGF-R ELISA (EGFR) gene, specifically sees Table 1 mutually:
Table 1: 29 kinds of sudden changes of patients with lung cancer EGF-R ELISA (EGFR) gene
29 kinds of sudden changes divide 8 PCR reaction detection, and the sudden change that each pipe detects sees Table 2.
Described Auele Specific Primer sees Table 3 mutually with specific probe.
Table 3 Auele Specific Primer and specific probe
Described a kind of fluorescence PCR method that detects 29 kinds of sudden changes of human epidermal growth factor acceptor EGFR gene does not comprise to be handled and the template extraction step testing sample, but for still having amplification and the detectivity same with the flesh tissue sample DNA from the paraffin-embedded sample of formaldehyde fixed with from the short segment DNA that sample obtained of blood plasma.
The invention has the beneficial effects as follows: the present invention is on the basis of Auele Specific Primer and dicyclo probe technique, set up multiple PCR in real time and detected 29 kinds of human epidermal growth factor acceptors (EGFR) transgenation simultaneously, this method: (1) can detect 19 deletion mutantions of EGFR gene simultaneously in a PCR pipe; (2) highly sensitive, the sudden change of 5-10 copy can detect; (3) high specificity, 10ng wild type gene group DNA does not have non-special signal; (4) the selectivity ability is strong, can detect 0.1% ~ 1% deletion mutantion DNA under 10ng wild type gene group DNA background; (5) detection speed is fast, and whole testing process needs 90 minutes.
Description of drawings
Fig. 1 to Fig. 2 detects the detection curve figure of 19 kinds of EGFR genetically deficient sudden changes for single tube fluorescent PCR of the present invention.
Fig. 1 does not contain the negative control sample detection graphic representation of 19 kinds of EGFR genetically deficient sudden changes for single tube fluorescent PCR of the present invention detects;
Fig. 2 contains wherein one or more the sample detection graphic representation of 19 kinds of EGFR genetically deficient sudden changes for single tube fluorescent PCR of the present invention detects;
Embodiment
The present invention is a detected object with 19 kinds of deletion mutantions of human EGFR gene 19 exons, optimum combination by special primer and unite and use the dicyclo probe, thereby realize detecting simultaneously quick and precisely, simply 19 kinds of deletion mutantions, and the ability of detection sudden change is up to 0.1% ~ 0.1%.
Wild-type cell is that the genomic dna of H460 is the wild-type template, with clone H1650 is 19-M1 (2235_2249del15) template, other 18 kinds of deletion mutantions with the mutant plasmid that makes up through genetically engineered as the sudden change template, set up the reaction system that the single tube fluorescent PCR detects 19 kinds of deletion mutantions of EGFR gene, and this system is used for the rapid detection of EGFR deletion mutantion.This method mainly may further comprise the steps:
(1) wild type gene sequence and 19 kinds of deletion mutantion gene orders of human epidermal growth factor acceptor (EGFR) gene 19 exons of announcing according to the Cosmic data design many to high specific degenerated primer and a plurality of specificity dicyclo probe.
(2) testing sample is handled and template extraction.
(3) fluorescent PCR amplification.
(4) detect fluorescent signal, the standard that needed cycle index Ct value is as a result of judged when reaching preset threshold.
Wild type gene sequence and 19 kinds of deletion mutantion gene orders of human epidermal growth factor acceptor (EGFR) gene 19 exons of announcing according to the Cosmic data in the step (1), design manyly, see table 3 for details high specific degenerated primer and a plurality of specificity dicyclo probe.
Step (2) sample preparation and template extraction, the sample scope of application comprise the fresh pathological tissue of excision, formaldehyde fixed paraffin embedding pathological anatomy, paraffin section, whole blood, blood plasma, serum, samples such as hydrothorax.Fresh pathological tissue is got the mung bean size, and about 1g is heavy, uses Qiagen company organization DNA extraction test kit to extract genomic dna, and concrete steps are pressed the test kit operation instructions.The wax stone sample is cut into 5～8 μ m section, gets 5, or 5～8 μ m sheets of having made get 5, after the dimethylbenzene dewaxing, uses Qiagen company paraffin embedding DNA extraction test kit to extract genomic dna, and concrete steps are pressed the test kit operation instructions.Whole blood, blood plasma, serum and hydrothorax sample use Qiagen company organization DNA extraction test kit to extract genomic dna, and concrete steps are pressed the test kit operation instructions.Each whole blood 200 μ l that extract, blood plasma, serum and hydrothorax are no less than 800 μ l.The DNA that carries is dissolved in Tris-HCl (10mmol/L, pH 8.0), extracts quality through UV spectrophotometer measuring, and definite concentration, adjusts DNA concentration to the template of 2ng/ μ l as pcr amplification with Tris-HCl solution (10mmol/L, pH 8.0).
The fluorescent PCR amplification reaction system of step (3) is:
1 * PCR damping fluid
MgCl
2 7.0mmol
Each 1.0mmol of dNTP
Each is to primer 0.1 ~ 1.0 μ mol
Each bar probe 0.5 ~ 1.0 μ mol
Taq enzyme 2.5U
Template 5 μ l
Above reagent component: 1 * PCR damping fluid, MgCl
2, Taq enzyme, dNTP are all available from the precious biotech firm of DaLian, China.
The reaction conditions of described fluorescent PCR is 95 ℃ of pre-sex change 5 minutes, 94 ℃ of sex change of 15 circulations 15 seconds, annealed 40 seconds for 66 ℃, 72 ℃ were extended 10 seconds, 94 ℃ of sex change of 35 circulations 15 seconds were annealed 40 seconds for 56 ℃, and 72 ℃ were extended 10 seconds, 35 circulations, back 35 circulate in detection FAM and HEX or ROX fluorescent signal when annealing.
Step (4) detects the Ct value of FAM and HEX fluorescent signal, is to adopt Mx3000P fluorescent PCR amplification instrument or Mx3005P fluorescent PCR amplification instrument or Rotor-Gene3000 fluorescent PCR amplification instrument or Rotor-Gene6000 fluorescent PCR amplification instrument or ABI 7500 fluorescent PCRs amplification instrument., once can detect 96 duplicate samples (comprising the yin and yang attribute contrast).Ct value judged result according to the demonstration of fluorescent PCR amplification instrument: the FAM of detection reaction system and HEX fluorescence intensity, show sample DNA amount when reaching setting threshold (Ct＞18) in allowed band with the HEX signal, the FAM signal results is credible; The standard that needed cycle index Ct value is judged as yin and yang attribute when reaching preset threshold with FAM, the Ct value is 0 or 35: feminine gender; Ct is less than 32: the positive.
Embodiment 1
Sport example with the E746_A750del (2235_2249del15) on the EGFR gene hot sudden change exons 19 in this example and analyze the method that single tube fluorescent PCR of the present invention detects 19 kinds of deletion mutantions of EGFR gene.Experiment is respectively H1650 (band E746_A750del sudden change), H460 (EGFR gene wild-type), 293T (EGFR gene wild-type), SW480 (EGFR gene wild-type) with clone 4 strains; The Voluntary Blood Donors whole blood sample of 100 parts of health, 62 parts of clinical lung cancer samples (comprising flesh tissue, paraffin section, hydrothorax, whole blood).
The method of utilizing above-mentioned fluorescent PCR to detect E746_A750del (2235_2249del15) sudden change on the EGFR gene hot sudden change exons 19 may further comprise the steps:
(1) sample preparation and template extraction Quality Control:
The sample scope of application comprises the fresh pathological tissue of excision, formaldehyde fixed paraffin embedding pathological anatomy, paraffin section, whole blood, blood plasma, serum, samples such as hydrothorax.Fresh pathological tissue is got the mung bean size, and about 1g is heavy, uses Qiagen company organization DNA extraction test kit to extract genomic dna, and concrete steps are pressed the test kit operation instructions.The wax stone sample is cut into 5～8 μ m section, gets 5, or 5～8 μ m that made section gets 5, after the dimethylbenzene dewaxing, uses Qiagen company paraffin embedding DNA extraction test kit to extract genomic dna, and concrete steps are pressed the test kit operation instructions.Whole blood, blood plasma, serum and hydrothorax sample use Qiagen company organization DNA extraction test kit to extract genomic dna, and concrete steps are pressed the test kit operation instructions.Each whole blood 200 μ l that extract, blood plasma, serum and hydrothorax are no less than 800 μ l.The DNA that carries is dissolved in Tris-HCl (10mmol/L, pH 8.0), extract quality through UV spectrophotometer measuring, and definite concentration, adjust DNA concentration to 100ng/ μ l or 2ng/ μ l template with Tris-HCl solution (10mmol/L, pH 8.0) as pcr amplification.
(2) fluorescent PCR amplification, the preparation reaction system:
1 * PCR damping fluid
MgCl
2 7.0mmol
Each 1.0mmol of dNTP
Each is to primer 0.1 ~ 1.0 μ mol
Each bar probe 0.5 ~ 1.0 μ mol
Taq enzyme 2.5U
Template 5 μ l
Cumulative volume 50 μ l
The reaction conditions of described fluorescent PCR is 95 ℃ of pre-sex change 5 minutes, and 94 ℃ of sex change of 15 circulations 15 seconds were annealed 40 seconds for 66 ℃, and 72 ℃ were extended 10 seconds, and 94 ℃ of sex change of 35 circulations 15 seconds were annealed 40 seconds for 56 ℃, and 72 ℃ were extended 35 circulations 10 seconds.
(3) detect: adopt back 35 cycle annealing stages of Mx3000P PCR in real time amplification instrument (StrataGene company) to detect FAM and HEX or ROX fluorescent signal, once can detect 96 duplicate samples (comprising the yin and yang attribute contrast).The result judges: the Ct value judged result that shows according to fluorescent PCR amplification instrument: the FAM of detection reaction system and HEX fluorescence intensity, show when reaching setting threshold (Ct＞18) that sample DNA measures in allowed band with the HEX signal, and the FAM signal results is credible; The standard that needed cycle index Ct value is judged as yin and yang attribute when reaching preset threshold with FAM, the Ct value is 0 or 35: feminine gender; Ct is less than 32: the positive.
Aforementioned 100 parts of healthy blood donor's whole blood samples and clone H460 (EGFR gene wild-type), 293T (EGFR gene wild-type), SW480 (EGFR gene wild-type), mutational cell line H1650 (band E746_A750del sudden change) detects through system of the present invention, have only the H1650 cell line dna that fluorescent signal is arranged, other samples do not have fluorescent signal, have further proved the specificity of fluorescent PCR.
Sensitivity analysis: with mutant cell be H1650DNA from the continuous 10 times of gradient dilutions of 100ng/ μ L, be respectively 100ng/ μ L, 10ng/ μ L, 1ng/ μ L, 100pg/ μ L, 10pg/ μ L, 1pg/ μ L.Each reaction adds 5 μ L DNA.The result shows the highly sensitive of fluorescence PCR method of the present invention, and 5 copy DNA genomes can detect.
The selectivity capability analysis: fixing each PCR reacts total DNA consumption, 100ng/ reaction and 10ng/ reaction.DNA and wild-type cell (H460) DNA of system concentration with mutational cell line (H1650) all is adjusted into 20ng/ μ L and 2ng/ μ L earlier.Each reaction adds that 5 μ L templates are 100ng/ reaction and 10ng/ reacts like this.Dispose analog D NA template as follows.
A:50% is 100ng/ μ L mutant cell DNA.
B:30% sneaks into 40 μ L 100ng/ μ L H460 cell DNAs after getting A liquid 60 μ L, and concussion is mixed.
C:20% sneaks into 60 μ L 100ng/ μ L H460 cell DNAs after getting A liquid 40 μ L, and concussion is mixed.
D:15% sneaks into 50 μ L 100ng/ μ L H460 cell DNAs after getting B liquid 50 μ L, and concussion is mixed.
E:10% sneaks into 50 μ L 100ng/ μ L H460 cell DNAs after getting C liquid 50 μ L, and concussion is mixed.
F:5% sneaks into 50 μ L 100ng/ μ L H460 cell DNAs after getting E liquid 50 μ L, and concussion is mixed.
G:1% sneaks into 80 μ L 100ng/ μ L H460 cell DNAs after getting F liquid 20 μ L, and concussion is mixed.
H:0.5% sneaks into 50 μ L 100n/ μ L H460 cell DNAs after getting G liquid 50 μ L, and concussion is mixed.
I:0.1% sneaks into 80 μ L 100n/ μ L H460 cell DNAs after getting H liquid 20 μ L, and concussion is mixed.
The result shows the selectivity detectivity of fluorescence PCR method of the present invention for can detect the mutant DNA of 5 copies in the total DNA of 10ng, and detectivity is 0.1%.
Replica test: each reaction adds mutational cell line H1650DNA10ng, 1ng and 100pg respectively, repeats 10 times and carries out the fluorescent PCR amplification, and 10 times the Ct value differs less than 0.1 circulation.
Collect the lung cancer specimen of excision, whole blood and plasma specimen and hydrothorax sample be totally 62 examples, 49 tissue samples wherein, 1 routine blood plasma, 3 routine hydrothorax, 9 routine whole bloods.The male sex's 34 examples wherein, women's 28 examples.Age is 36-71 year, and the mean age is 55 years old.
For exons 19, detected result of the present invention and dna sequencing result are in full accord: have 3 examples that the E746-A750 sudden change of exons 19 takes place in the 62 routine samples, the contrast of 15 routine normal lung tissues is wild-type.Wherein be 2 routine women, the 1 routine male sex, the women and the male sex have accounted for 67% and 33% respectively.
Table 4 fluorescent PCR detected result and dna sequencing result's comparison
The single PCR of the present invention reaction just can detect 19 kinds of disappearances of EGFR exons 19 simultaneously, and detection time is need 100 minutes only, so the present invention is time saving and energy saving, and the accuracy height can satisfy the quick diagnosis of sudden change.And fluorescence PCR method and traditional sequence measurement result's coincidence rate is 100%, and fluorescent PCR sensitivity and selectivity detectivity are higher than traditional sequence measurement, and containing 0.1% mutant DNA in the 10ng sample DNA can detect.
Embodiment 2
Sport example with the L747_P753＞S (2240_2257del18) on the EGFR gene hot sudden change exons 19 in this example and analyze the method that single tube fluorescent PCR of the present invention detects 19 kinds of deletion mutantions of EGFR gene.Test with plasmid template 1 strain (containing L747_P753＞S sudden change), clone 3 strains are respectively H460 (EGFR gene wild-type), 293T (EGFR gene wild-type), SW480 (EGFR gene wild-type); The Voluntary Blood Donors whole blood sample of 100 parts of health, 62 parts of clinical lung cancer samples (comprising flesh tissue, paraffin section, hydrothorax, whole blood).
The method of utilizing above-mentioned fluorescent PCR to detect L747_P753＞S (2240_2257del18) sudden change on the EGFR gene hot sudden change exons 19 may further comprise the steps:
(1) sample preparation and template extraction Quality Control:
The sample scope of application comprises the fresh pathological tissue of excision, formaldehyde fixed paraffin embedding pathological anatomy, paraffin section, whole blood, blood plasma, serum, samples such as hydrothorax.Fresh pathological tissue is got the mung bean size, and about 1g is heavy, uses Qiagen company organization DNA extraction test kit to extract genomic dna, and concrete steps are pressed the test kit operation instructions.The wax stone sample is cut into 5～8 μ m section, gets 5, or 5～8 μ m that made section gets 5, after the dimethylbenzene dewaxing, uses Qiagen company paraffin embedding DNA extraction test kit to extract genomic dna, and concrete steps are pressed the test kit operation instructions.Whole blood, blood plasma, serum and hydrothorax sample use Qiagen company organization DNA extraction test kit to extract genomic dna, and concrete steps are pressed the test kit operation instructions.Each whole blood 200 μ l that extract, blood plasma, serum and hydrothorax are no less than 800 μ l.The DNA that carries is dissolved in Tris-HCl (10mmol/L, pH 8.0), extract quality through UV spectrophotometer measuring, and definite concentration, adjust DNA concentration to 100ng/ μ l or 2ng/ μ l template with Tris-HCl solution (10mmol/L, pH 8.0) as pcr amplification.
(2) fluorescent PCR amplification, the preparation reaction system:
1 * PCR damping fluid
MgCl
2 7.0mmol
Each 1.0mmol of dNTP
Each is to primer 0.1 ~ 1.0 μ mol
Each bar probe 0.5 ~ 1.0 μ mol
Taq enzyme 2.5U
Template 5 μ l
Cumulative volume 50 μ l
The reaction conditions of described fluorescent PCR is 95 ℃ of pre-sex change 5 minutes, and 94 ℃ of sex change of 15 circulations 15 seconds were annealed 40 seconds for 66 ℃, and 72 ℃ were extended 10 seconds, and 94 ℃ of sex change of 35 circulations 15 seconds were annealed 40 seconds for 56 ℃, and 72 ℃ were extended 35 circulations 10 seconds.
(3) detect: adopt back 35 cycle annealing stages of Mx3000P PCR in real time amplification instrument (StrataGene company) to detect FAM and HEX or ROX fluorescent signal, once can detect 96 duplicate samples (comprising the yin and yang attribute contrast).The result judges: the Ct value judged result that shows according to fluorescent PCR amplification instrument: the FAM of detection reaction system and HEX fluorescence intensity, show when reaching setting threshold (Ct＞18) that sample DNA measures in allowed band with the HEX signal, and the FAM signal results is credible; The standard that needed cycle index Ct value is judged as yin and yang attribute when reaching preset threshold with FAM, the Ct value is 0 or 35: feminine gender; Ct is less than 32: the positive.
Aforementioned 100 parts of healthy blood donor's whole blood samples and clone H460 (EGFR gene wild-type), 293T (EGFR gene wild-type), SW480 (EGFR gene wild-type), mutant plasmid DNA (band L747_P753＞S sudden change) detects through system of the present invention, have only mutant plasmid DNA that fluorescent signal is arranged, other samples do not have fluorescent signal, have further proved the specificity of fluorescent PCR.
Sensitivity analysis: with the continuous 10 times of gradient dilutions of mutant plasmid DNA, each reaction adds 5 μ L DNA.The result shows the highly sensitive of fluorescence PCR method of the present invention, and 5 copy DNA genomes can detect.
The selectivity capability analysis: fixing each PCR reacts total DNA consumption, 100ng/ reaction and 10ng/ reaction.Earlier wild-type cell (H460) DNA of system concentration all is adjusted into 20ng/ μ L and 2ng/ μ L.Each reaction adds that 5 μ L templates are 100ng/ reaction and 10ng/ reacts like this.Then with 10
5The mutant plasmid DNA of the copy/μ L continuous 10 times of dilutions of H460DNA of 20ng/ μ L and 2ng/ μ L.
The result shows the selectivity detectivity of fluorescence PCR method of the present invention for can detect the mutant DNA of 5 copies in the total DNA of 10ng, and detectivity is 0.1%.
Replica test: each reaction adds mutant plasmid DNA1000 copy, 100 copies and 10 copies respectively, repeats 10 times and carries out the fluorescent PCR amplification, and 10 times the Ct value differs less than 0.1 circulation.
Collect the lung cancer specimen of excision, whole blood and plasma specimen and hydrothorax sample be totally 62 examples, 49 tissue samples wherein, 1 routine blood plasma, 3 routine hydrothorax, 9 routine whole bloods.The male sex's 34 examples wherein, women's 28 examples.Age is 36-71 year, and the mean age is 55 years old.
For exons 19, detected result of the present invention and dna sequencing result are in full accord: have 3 examples that the deletion mutantion of exons 19 takes place in the 62 routine samples, the contrast of 15 routine normal lung tissues is wild-type.Wherein be 2 routine women, the 1 routine male sex, the women and the male sex have accounted for 67% and 33% respectively.
Table 4 fluorescent PCR detected result and dna sequencing result's comparison
The single PCR of the present invention reaction just can detect 19 kinds of disappearances of EGFR exons 19 simultaneously, and detection time is need 100 minutes only, so the present invention is time saving and energy saving, and the accuracy height can satisfy the quick diagnosis of sudden change.And fluorescence PCR method and traditional sequence measurement result's coincidence rate is 100%, and fluorescent PCR sensitivity and selectivity detectivity are higher than traditional sequence measurement, and containing 0.1% mutant DNA in the 10ng sample DNA can detect.
Embodiment 3
Sport example with the E746_T751del (2236_2253del18) on the EGFR gene hot sudden change exons 19 in this example and analyze the method that single tube fluorescent PCR of the present invention detects 19 kinds of deletion mutantions of EGFR gene.Test with plasmid template 1 strain (containing the E746_T751del sudden change), clone 3 strains are respectively H460 (EGFR gene wild-type), 293T (EGFR gene wild-type), SW480 (EGFR gene wild-type); The Voluntary Blood Donors whole blood sample of 100 parts of health, 62 parts of clinical lung cancer samples (comprising flesh tissue, paraffin section, hydrothorax, whole blood).
The method of utilizing above-mentioned fluorescent PCR to detect L747_P753＞S (2240_2257del18) sudden change on the EGFR gene hot sudden change exons 19 may further comprise the steps:
(1) sample preparation and template extraction Quality Control:
The sample scope of application comprises the fresh pathological tissue of excision, formaldehyde fixed paraffin embedding pathological anatomy, paraffin section, whole blood, blood plasma, serum, samples such as hydrothorax.Fresh pathological tissue is got the mung bean size, and about 1g is heavy, uses Qiagen company organization DNA extraction test kit to extract genomic dna, and concrete steps are pressed the test kit operation instructions.The wax stone sample is cut into 5～8 μ m section, gets 5, or 5～8 μ m sheets of having made get 5, after the dimethylbenzene dewaxing, uses Qiagen company paraffin embedding DNA extraction test kit to extract genomic dna, and concrete steps are pressed the test kit operation instructions.Whole blood, blood plasma, serum and hydrothorax sample use Qiagen company organization DNA extraction test kit to extract genomic dna, and concrete steps are pressed the test kit operation instructions.Each whole blood 200 μ l that extract, blood plasma, serum and hydrothorax are no less than 800 μ l.The DNA that carries is dissolved in Tris-HCl (10mmol/L, pH 8.0), extract quality through UV spectrophotometer measuring, and definite concentration, adjust DNA concentration to 100ng/ μ l or 2ng/ μ l template with Tris-HCl solution (10mmol/L, pH 8.0) as pcr amplification.
(2) fluorescent PCR amplification, the preparation reaction system:
1 * PCR damping fluid
MgCl
2 7.0mmol
Each 1.0mmol of dNTP
Each is to primer 0.1 ~ 1.0 μ mol
Each bar probe 0.5 ~ 1.0 μ mol
Taq enzyme 2.5U
Template 5 μ l
Cumulative volume 50 μ l
The reaction conditions of described fluorescent PCR is 95 ℃ of pre-sex change 5 minutes, and 94 ℃ of sex change of 15 circulations 15 seconds were annealed 40 seconds for 66 ℃, and 72 ℃ were extended 10 seconds, and 94 ℃ of sex change of 35 circulations 15 seconds were annealed 40 seconds for 56 ℃, and 72 ℃ were extended 35 circulations 10 seconds.
(3) detect: adopt back 35 cycle annealing stages of Mx3000P PCR in real time amplification instrument (StrataGene company) to detect FAM and HEX or ROX fluorescent signal, once can detect 96 duplicate samples (comprising the yin and yang attribute contrast).The result judges: the Ct value judged result that shows according to fluorescent PCR amplification instrument: the FAM of detection reaction system and HEX fluorescence intensity, show when reaching setting threshold (Ct＞18) that sample DNA measures in allowed band with the HEX signal, and the FAM signal results is credible; The standard that needed cycle index Ct value is judged as yin and yang attribute when reaching preset threshold with FAM, the Ct value is 0 or 35: feminine gender; Ct is less than 32: the positive.
Aforementioned 100 parts of healthy blood donor's whole blood samples and clone H460 (EGFR gene wild-type), 293T (EGFR gene wild-type), SW480 (EGFR gene wild-type), mutant plasmid DNA (band L747_P753＞S sudden change) detects through system of the present invention, have only mutant plasmid DNA that fluorescent signal is arranged, other samples do not have fluorescent signal, have further proved the specificity of fluorescent PCR.
Sensitivity analysis: with the continuous 10 times of gradient dilutions of mutant plasmid DNA.Each reaction adds 5 μ L DNA.The result shows the highly sensitive of fluorescence PCR method of the present invention, and 5 copy DNA genomes can detect.
The selectivity capability analysis: fixing each PCR reacts total DNA consumption, 100ng/ reaction and 10ng/ reaction.Earlier wild-type cell (H460) DNA of system concentration all is adjusted into 20ng/ μ L and 2ng/ μ L.Each reaction adds that 5 μ L templates are 100ng/ reaction and 10ng/ reacts like this.Then with 10
5The mutant plasmid DNA of the copy/μ L continuous 10 times of dilutions of H460DNA of 20ng/ μ L and 2ng/ μ L.
The result shows the selectivity detectivity of fluorescence PCR method of the present invention for can detect the mutant DNA of 5 copies in the total DNA of 10ng, and detectivity is 0.1%.
Replica test: each reaction adds mutant plasmid DNA1000 copy, 100 copies and 10 copies respectively, repeats 10 times and carries out the fluorescent PCR amplification, and 10 times the Ct value differs less than 0.1 circulation.
Collect the lung cancer specimen of excision, whole blood and plasma specimen and hydrothorax sample be totally 62 examples, 49 tissue samples wherein, 1 routine blood plasma, 3 routine hydrothorax, 9 routine whole bloods.The male sex's 34 examples wherein, women's 28 examples.Age is 36-71 year, and the mean age is 55 years old.
For exons 19, detected result of the present invention and dna sequencing result are in full accord: have 3 examples that the deletion mutantion of exons 19 takes place in the 62 routine samples, the contrast of 15 routine normal lung tissues is wild-type.Wherein be 2 routine women, the 1 routine male sex, the women and the male sex have accounted for 67% and 33% respectively.
Table 4 fluorescent PCR detected result and dna sequencing result's comparison
The single PCR of the present invention reaction just can detect 19 kinds of disappearances of EGFR exons 19 simultaneously, and detection time is need 100 minutes only, so the present invention is time saving and energy saving, and the accuracy height can satisfy the quick diagnosis of sudden change.And fluorescence PCR method and traditional sequence measurement result's coincidence rate is 100%, and fluorescent PCR sensitivity and selectivity detectivity are higher than traditional sequence measurement, and containing 0.1% mutant DNA in the 10ng sample DNA can detect.
Sequence table
＜110〉Xiamen Amoydx Bio-Pharmaceutical Technology Co., Ltd.
＜120〉be used to detect primer, probe and the using method thereof that human EGFR gene suddenlys change
<160>24
<210>1
<211>26
<212>DNA
＜213〉artificial sequence
<400>1
GAATTCAAAA?AGATCAAAGT?GCTGGC 26
<210>2
<211>27
<212>DNA
＜213〉artificial sequence
<400>2
CTGAATTCAA?AAAGATCAAA?GTGCTGA 27
<210>3
<211>26
<212>DNA
＜213〉artificial sequence
<400>3
CTGAATTCAA?AAAGATCAAA?GTGCTGT 26
<210>4
<211>19
<212>DNA
＜213〉artificial sequence
<400>4
TCTGGGCTCC?CCACCAGAC 19
<210>5
<211>26
<212>DNA
＜213〉artificial sequence
<400>5
GTTAAAATTC?CCGTCGCTAT?CAAAAC 26
<210>6
<211>29
<212>DNA
＜213〉artificial sequence
<400>6
GTTAAAATTC?CCGTCGCTAT?CAAAATATC 29
<210>7
<211>20
<212>DNA
＜213〉artificial sequence
<400>7
TCCCGTCGCT?ATCAAGGATC 20
<210>8
<211>19
<212>DNA
＜213〉artificial sequence
<400>8
CCCGTCGCTA?TCAAGGAGC 19
<210>9
<211>20
<212>DNA
＜213〉artificial sequence
<400>9
TCCCGTCGCT?ATCAAGGAAC 20
<210>10
<211>24
<212>DNA
＜213〉artificial sequence
<400>10
TTCCCGTCGC?TATCAAGGAA?TCTT 24
<210>11
<211>22
<212>DNA
＜213〉artificial sequence
<400>11
CGGCTGCCAG?ACATGAGAAAAG 22
<210>12
<211>20
<212>DNA
＜213〉artificial sequence
<400>12
GAAGCCTACG?TGATGGCCAT 20
<210>13
<211>18
<212>DNA
＜213〉artificial sequence
<400>13
GTGGACAACC?CCCACCAC 18
<210>14
<211>17
<212>DNA
＜213〉artificial sequence
<400>14
CGTGGACAAC?CCCCACC 17
<210>15
<211>18
<212>DNA
＜213〉artificial sequence
<400>15
ATGGCCAGCG?TGGACGGT 18
<210>16
<211>16
<212>DNA
＜213〉artificial sequence
<400>16
TGATGGCCAG?CGTGTC 16
<210>17
<211>18
<212>DNA
＜213〉artificial sequence
<400>17
GAGCAGGTAC?TGGGAGCC 18
<210>18
<211>24
<212>DNA
＜213〉artificial sequence
<400>18
TGTCAAGATC?ACAGATTTTG?GGGG 24
<210>19
<211>22
<212>DNA
＜213〉artificial sequence
<400>19
CAGATTTTGG?GCTGGCCAAA?AA 22
<210>20
<211>23
<212>DNA
＜213〉artificial sequence
<400>20
GGAAAATGCT?GGCTGACCTA?AAG 23
<210>21
<211>35
<212>DNA
＜213〉artificial sequence
<400>21
CGTGCCGCGT?TCGGCACGGT?GTATAAGGAC?ACCGT 35
<210>22
<211>34
<212>DNA
＜213〉artificial sequence
<400>22
TTCTGCTAAA?GCAGAAACTC?ACATCGAGAT?GTGA 34
<210>23
<211>31
<212>DNA
＜213〉artificial sequence
<400>23
AGCCGACCTT?CGGCTGCCTC?CTGGAAGGAG?G 31
<210>24
<211>39
<212>DNA
＜213〉artificial sequence
<400>24
AAGTAAGCCT?CCTTACTTTG?CCTCCTTCTG?CAAGGAGGC 39
Claims (5)
1, be used to detect the primer and the probe of human EGFR gene sudden change, comprise at least one group in following 4 groups of primers and the probe:
(1)、5′-GAATTCAAAAAGATCAAAGTGCTGGC-3′SEQ?ID?NO：1
5′-CTGAATTCAAAAAGATCAAAGTGCTGA-3′SEQ?ID?NO：2
5′-CTGAATTCAAAAAGATCAAAGTGCTGT-3′SEQ?ID?NO：3
5′-TCTGGGCTCCCCACCAGAC-3′SEQ?ID?NO：4
FAM-5′-CGTGCCGCGTTCGGCACGGTGTATAAGGACACCGT-3′-TAMRA?SEQ?ID?NO：21
(2)、5′-GTTAAAATTCCCGTCGCTATCAAAAC-3′SEQ?ID?NO：5
5′-GTTAAAATTCCCGTCGCTATCAAAATATC-3′SEQ?ID?NO：6
5′-TCCCGTCGCTATCAAGGATC-3′SEQ?ID?NO：7
5′-CCCGTCGCTATCAAGGAGC-3′SEQ?ID?NO：8
5′-TCCCGTCGCTATCAAGGAAC-3′SEQ?ID?NO：9
5′-TTCCCGTCGCTATCAAGGAATCTT-3′SEQ?ID?NO：10
5′-CGGCTGCCAGACATGAGAAAAG-3′SEQ?ID?NO：11
FAM-5′TTCTGCTAAAGCAGAAACTCACATCGAGATGTGA-3′-TAMRA?SEQ?ID?NO：22
(3)、5′-GAAGCCTACGTGATGGCCAT-3′SEQ?ID?NO：12
5′-GTGGACAACCCCCACCAC-3′SEQ?ID?NO：13
5′-CGTGGACAACCCCCACCA-3′SEQ?ID?NO：14
5′-ATG?GCCAGCGTGGACGGT-3′SEQ?ID?NO：15
5′-TGATGGCCAGCGTGTC-3′SEQ?ID?NO：16
5′-GAGCAGGTACTGGGAGCC-3′SEQ?ID?NO：17
FAM-5′AGCCGACCTTCGGCTGCCTCCTGGAAGGAGG-3′-TAMRA?SEQ?ID?NO：23
(4)、5′TGTCAAGATCACAGATTTTGGGGG-3′SEQ?ID?NO：18
5′CAGATTTTGGGCTGGCCAAAAA-3′SEQ?ID?NO：19
5′-GGAAAATGCTGGCTGACCTAAAG-3′SEQ?ID?NO：20
FAM-5′-AAGTAAGCCTCCTTACTTTGCCTCCTTCTGCAAGGAGGC-3′-TAMRA?SEQ?ID?NO：24
2, a kind of method that detects the human EGFR gene sudden change may further comprise the steps:
(1) provides in described 4 groups of primers of claim 1 and the probe at least one group;
(2) testing sample is handled and template extraction;
(3) reaction system of preparation fluorescent PCR amplification mutator gene sequence;
(4) primer and the probe amplification mutator gene target sequence to be measured of usefulness step (1);
(5) utilize the hybridization of dicyclo probe and amplified production, the FAM of detection reaction system and HEX or ROX fluorescence intensity show sample DNA amount in allowed band when reaching setting threshold (Ct＞18) with HEX (or ROX) signal, and the FAM signal results is credible; The standard that needed cycle index Ct value is judged as yin and yang attribute when reaching preset threshold with FAM, the Ct value is 0 or 35: feminine gender; Ct is less than 32: the positive.
3, a kind of method that detects the human EGFR gene sudden change as claimed in claim 2, it is characterized in that: the described testing sample of step (2) comprises the fresh pathological tissue of excision, formaldehyde fixed paraffin embedding pathological anatomy, paraffin section, whole blood, blood plasma, serum, hydrothorax.
4, a kind of method that detects the human EGFR gene sudden change as claimed in claim 2, wherein the reaction system of fluorescent PCR is:
1 * PCR damping fluid
MgCl
2 5.0～10.0mmol
Each 0.8～1.5mmol of dNTP
Each is to primer 0.1～1.0 μ mol
Each bar probe 0.5～1.0 μ mol
Taq enzyme 2.0～3.0U
Template 4.0～6.0 μ l
Cumulative volume 40～60 μ l
5, a kind of test kit comprises at least:
In described 4 groups of primers of claim 1 and the probe at least one group;
And PCR reaction buffer.
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