CN101586153A - Kit for forecasting treatment effect of epidermal growth factor receptor inhibitor - Google Patents

Abstract

The invention relates to a kit for forecasting the treatment effect of an epidermal growth factor receptor inhibitor, in particular to a specific primer and a probe for detecting the polymorphism of a nineteenth exon of an epidermal growth factor receptor gene by a PCR method, application of the same in preparing a reagent for detecting the polymorphism of the nineteenth exon of the epidermal growth factor receptor gene through a PCR amplified biological nucleic acid sample, a kit for detecting the polymorphism of the nineteenth exon of the epidermal growth factor receptor gene and application of the kit, and belongs to the field of medicaments.

Description

The test kit of prediction epidermal growth factor receptor inhibitor curative effect

Technical field

The present invention relates to by detecting the gene pleiomorphism of EGF-R ELISA, the curative effect of prediction growth factor receptor inhibitor medicine.The invention belongs to field of medicaments.

Background technology

Pharmacogenomics is mainly studied different patients due to the genovariation to the differential responses of medicine, and develops new medicine or new administrated method on this basis.Pharmacogenomics is different from genetics in general sense, and it is not finding that human genome is a main purpose, but improves patient's treatment with known genetics theory simply relatively.Pharmacogenomics can not be regarded the easily ill physique of prediction simply as, perhaps provide statistical information to be used to predict the test of onset risk, its influence that to be the research related gene produce drug effect itself and with the relation of genovariation, and these variations are basic reasons of Different Individual generation different pharmaceutical effect.Pharmacogenomics is a purpose to pursue drug effect and security, the relation between research range gene sudden change and drug effect and the security.Mainly in the difference of gene level research drug effect, because the development of gene analysis technique, pharmacogenomics can be increasing to pharmaceutical industries for pharmacogenomics.

(Epidermal Growth Factor Receptor, EGFR) gene is positioned at the short arm of a chromosome 7p12-14 district No. 7 to EGF-R ELISA, is made up of 28 exons.Coded protein belongs to the I type and strides film Tyrosylprotein kinase growth factor receptors, and molecular weight is about 170KDa.The precursor of EGFR is the single polypeptide chain that contains 1210 amino-acid residues, and wherein 24 amino acid are the signal peptide section, and are cleaved when translation process, so the receptor tyrosine protein kinase that sophisticated EGFR is made up of 1186 amino acid.EGFR comprises extracellular region, strides the film district, three parts of intracellular region, and the single a small amount of amino-acid residue of striding film sequence and these sequence both sides of exons 15 codings is striden the film district and is made up of 23 amino-acid residues, and acceptor is anchored on the after birth.The extracellular region of EGFR is encoded by exon 2-14.Being made up of 621 amino acid, is that it combines the position, place with corresponding part.The Tyrosylprotein kinase district is made up of 542 amino-acid residues exon, is typical A TP binding site and Tyrosylprotein kinase district.

EGFR is distributed widely in normal Mammals surface epithelial cell, and on average each cell receptor number is 5 * 10 ⁴-10 * 10 ⁴Studies show that the EGFR part by interacting with EGFR, transmits the mitotic division signal downwards, thereby cell cycle regulation, regulate the cell growth with differentiation with promote injury repairing.In the healthy tissues, EGFR is low to express or does not express, and EGFR was expression in comprising the multiple epithelium source property tumour of nonsmall-cell lung cancer, and descended in close relationsly with tumor proliferative, transfer, chemicotherapy susceptibility, was the gene therapy target position that has development prospect.

Discovering to lung cancer, mammary cancer, ovarian cancer, cervical cancer, prostate cancer, bladder cancer, colorectal carcinoma, the esophageal carcinoma, lung cancer, tumor of head and neck and cerebral tumor, the tumour of EGFR signal conduction imbalance accounts for quite great proportion (Kim HG, [J] .Histol Histopathol, 1999,14 (4): 1175-1182).The high expression level of EGFR can promote apoptosis (NormannoN, J Cell Physiol, 2003,194 (1): 13-19) of propagation, tumor-blood-vessel growth, adhesion, invasion and attack, transfer and the tumour cell of tumour cell.

Target inhibitor at EGFR mainly contains 4 classes: the monoclonal antibody (EGFR-Mab) of 1. blocking the outer receptor functional domain of EGFR born of the same parents; 2. the small-molecule drug (EGFR-TKI) that suppresses Tyrosylprotein kinase (TK) functional zone autophosphorylation in the EGFR born of the same parents; 3. block the antisense nuclear former times acid that EGFR produces; 4. some toxin and EGFR and ligand thereof or monoclonal antibody form mixture, reach the purpose of kill tumor cell by its toxicity.Wherein, small molecules tyrosine kinase inhibitor (EGFR-TKI) with Gefitinib (English name Gefitinib, trade(brand)name: Iressa Iressa) and erlotinib (English name: Erlotinib, trade(brand)name: Te Luokai Tarceva) for representative.

The clinical trial of II phase IDEAL1 and IDEAL2 shows, the efficient of nonsmall-cell lung cancer in late period (NSCLC) patient of ZD1839 single therapy chemotherapy failure is respectively 18.4% and 11.8%, stable disease rate (SD) is respectively 54.4% and 42.2% (MuXL, [J] .Clin Cancer Res, 2005,11:4289-4294.Chabner BA.[J] .Oncologist, 2004,9:245-246).Discover that it mainly is the autophosphorylation that stops EGFR by the ATP-binding site of TK functional zone in the competitiveness blocking-up born of the same parents, and then through following approach improve radiosensitivity for tumor cell 1. EGFR-TKI can strengthen the cell cycle redistribution effect of radiotherapy-induced, make more cell concentrate on the G1-M phase, and S phase cell still less; 2. improve radiotherapy inductive apoptosis of tumor cells; 3. suppress the reparation of the tumour cell damage that radiotherapy causes.In addition, discover also that ZD1839 can also suppress the vasculogenesis in the tumour and further improve the radiosensitivity of tumour.

EGFR belongs to I type growth factor receptors family, has receptor tyrosine kinase activity.3 important structure are contained in the Tyrosylprotein kinase functional zone of EGFR: 1. aminoterminal leaflet (N-lobe), constitute by 5 parallel b-foldings, and the GSGSFG sequence of ATP phosphoric acid coupling collar (P-Loop) wherein is the main binding site of ATP γ-phosphate group; 2. α C spiral (α Chelix) contains the α of ATP-and β-phosphate group binding site in this spiral.When α C spiral with after ATP combines, its conformation changes, thereby causes receptor autophosphorylation phosphorylation and kinase activation; 3. carboxyl terminal leaflet (C-lobe), activation ring (A-loop) in this leaflet is the active centre of Tyrosylprotein kinase, be made up of 20～30 amino acid, wherein the DFG sequence is conservative at all Tyrosylprotein kinase camber, changes the composition of this sequence or conformation with the remarkably influenced kinase activity.

The EGFR transgenation of Fa Xianing up to now is positioned at exons 1 8～21 more than 90%, these sudden changes can be divided into 4 types:

1. 155 G of the 18th exon 2 → A displacement causes glycine 719 codon missense mutation to become halfcystine (G719S).

2. exons 19 base deletions mainly are the base deletion sudden changes of the 746th～752 bit codon, cause amino acid in the EGFR albumen (ELREATS) sequence to be lost, this disappearance changed acceptor ATP in conjunction with capsule (ATP-bindingpocket, angle ABP), thereby significantly strengthen the susceptibility (SodellaR of cancer cells to Gefitinib, .Science, 2004,305:1163-1167.KosakaT, Cancer Res, 2004,64:8919-8923.).According to incompletely statistics, exons 19 transgenations of having found both at home and abroad at present have 22 kinds of different types at least.

3. the sudden change of the point mutation of extron 20 or base insertion catastrophe point mainly is that the C-T conversion appears in the 790th bit codon, the amino acid that causes this site in the EGFR albumen changes methionine(Met) (T790M) into by Threonine, this sudden change is detected in recidivist after the pharmacological agent, sudden change makes cancer cells produce resistance (PaoW to Gefitinib and Erlotinib, PLoS Medicine, 2005,2:57-61).Base is inserted and is mutated present the 770th～775 bit codon, and there are 8 kinds of different inserted modes in (mainly in CCCCC sequence both sides) between the GACAACCCCCACGTGTGC sequence, and the fragment of insertion is 3～9 bases.The clinical meaning that this class is inserted sudden change it be not immediately clear (Shigmatsu H .JNCI, 2005,97:339-346).

4. the point mutation of exon 21 mainly is that the T-G conversion appears in the 858th bit codon, the amino acid that causes this site in the EGFR albumen changes arginine (being called for short L858R) into by leucine, this sudden change is positioned near the DFG sequence, its effect is that the stability of A-loop is improved, and cancer cells obviously strengthens the susceptibility of Gefitinib and Erlotinib.

(LynchTJ such as the Lynch of U.S. Harvard Medical School, NEnglJ Med, 2004,350:2129-2139.) reported that 16 examples accept the lung cancer patient of Gefitinib treatment, there is the EGFR transgenation in 8 examples (wherein 4 examples are del E746-A750 among the 9 routine responders, 2 examples are exon 21 L858R, 1 example is L861Q, in addition 1 example mutate present exons 18, be G719C, and 7 routine nonresponders are wild-type, disclose the relation of EGRF transgenation and curative effect of medication first.Shortly after that, (PaezJG such as another group investigator Paez of this institute, Science, 2004,304:1497-1500.) report that also in 9 routine lung cancer patients, 5 routine Gefitinib treatment responders are the EGFR mutant, and (4 examples are exons 19 deletion mutantions, 1 example is L858R), and 4 routine nonresponders are wild-type.(Pao WPNAS such as the Pao of Washington, DC university, 2004,101:13306-13311). reported similar result of study, (wherein 6 examples are exons 19 deletion mutantions to detect the EGFR transgenation in 10 routine Gefitinib treatment responder 7 examples, 1 example is L858R), and 8 routine nonresponders are wild-type.There is EGFR transgenation (3 examples are exons 19 deletion mutantions, and 3 examples are L858R, and wherein 1 example exists exons 19 and 21 sudden changes simultaneously) in the report but this author also takes the lead in, 7 examples 5 examples in Erotinib treatment responder, and 10 routine nonresponders are wild-type.

Except that American scholar, the Asia scholar has also reported result of study separately in succession.(HanSW such as the Han of Seoul, South Korea university, JClinOncol, 2005,23:1-9.) report, 17 routine EGFR mutant lung cancer are 11 routine produce effects 64.7% after the Gefitinib treatment, and wherein 4 examples are exons 19 deletion mutantions, and 5 examples are L858R, 2 examples are exons 18 sudden change G719A, and only 10 examples (13.7%) are effective in the 73 routine wild-type lung cancer.In addition, go back reported first, the progression of disease time (TTP) of EGFR mutant and overall survival rate indexs such as (OS) all significantly are better than wild-type, and prompting mutant patient is better through the prognosis of pharmacological agent.

Japan hospital of Aichi Cancer center (Mitsudomi J Clin Oncol, 2005,11:2513-2520). also reported the relation of EGFR transgenation, Gefitinib curative effect and prognosis.The author has analyzed the postoperative recurrence patient through Gefitinib treatment, find 20 example treatment back tumours dwindle 〉=30% patient in, 19 examples (95%) be the EGFR mutant, and after the 17 example treatments among the patient of disease progressions, only 2 examples (12%) are mutant.In addition, also observe, exons 19 base deletion persons' efficient (16/16 example, 100%) is significantly higher than point mutation (G719C, L858R and L861Q, 5/8 example, 67%), and treatment responder's survival rate is significantly higher than the nonresponder, and it is the principal element that influences prognosis that the Kaplan-Meier check discloses the EGFR transgenation.

(HuangSF such as TaiWan, China investigator Huang, ClinCancer Res, 2004,10:8195-8203.) at first reported the relation of China EGFR transgenation and Gefitinib treatment, accept among the patient of pharmacological agent in 19 examples, effective (PR) 6 examples are mutant, and (wherein 2 examples are exons 19 disappearances, 2 routine L858R, 2 examples are exon 21 sudden change A839T and K846R), and in invalid 7 examples only 1 example be mutant.

(MuXL such as Beijing consonance Mu of medical university, Clin Cancer Res, 2005,11:4289-4294.) reported the first part result of study of China's Mainland, 22 examples are 7 examples effectively (PR) in the patient of Gefitinib treatment, 7 routine stable disease (SD), 8 routine disease progressions (PD), in 14 routine patients PR+SD, 10 examples are mutant (71.4%), and patient PD is wild-type.With the report difference of Mitsudomi etc. be, 5 examples are PR in the 6 routine L858R sudden changes, and 1 example is SD, and 4 routine exons 19 lack in PR and SD respectively be 2 examples.

Summary of the invention

Technical problem to be solved by this invention is at the sudden change of prior art mesocuticle growth factor receptor gene, and a kind of probe that detects epidermal growth factor receptor gene the 19th exon polymorphism with PCR method is provided:

SEQUENCE 1：VIC-CCCAGAAGGTGAGAAAGTTAAAATTC；

SEQUENCE 2：FAM-CCCACACAGC。

Simultaneously, the present invention also provides a kind of Auele Specific Primer that detects epidermal growth factor receptor gene the 19th exon polymorphism with PCR method:

SEQUENCE 3：ATTAAGAGAAGCAACATCT；

SEQUENCE 4：CGCTATCAAAACATCT。

Simultaneously, the present invention provides Auele Specific Primer ATTAAGAGAAGCAACATCT, CGCTATCAAAACATCT and probe VIC-CCCAGAAGGTGAGAAAGTTAAAATTC, FAM-CCCACACAGC preparing by the purposes in the reagent of pcr amplification biotinylated nucleic acid sample detection epidermal growth factor receptor gene the 19th exon polymorphism again.Wherein, reagent comprises Taq archaeal dna polymerase, dNTP mixture, Mg ²⁺

Simultaneously, the present invention provides a kind of test kit that detects epidermal growth factor receptor gene the 19th exon polymorphism again, especially detects the test kit of epidermal growth factor receptor gene the 19th exon E746-A750del loci polymorphism.

Test kit provided by the invention comprises: polymerase chain reaction (PCR) amplification reagent composition, the detection composition, the epidermal growth factor receptor gene that detect the epidermal growth factor receptor gene polymorphism be wild/and mutator gene type positive control plasmid.

Wherein, the polymerase chain reaction (PCR) amplification reagent composition comprises Taq archaeal dna polymerase, polymerase chain reaction damping fluid, dNTP mixture, Mg ²⁺The ratio of each component is optimized through overtesting in the amplification.Wherein, the Taq archaeal dna polymerase, be can warm start the Taq archaeal dna polymerase.

Wherein, detect the detection composition of epidermal growth factor receptor gene polymorphism, comprising:

Probe:

SEQUENCE 1：VIC-CCCAGAAGGTGAGAAAGTTAAAATTC；

SEQUENCE 2：FAM-CCCACACAGC。

Primer:

SEQUENCE 3：ATTAAGAGAAGCAACATCT；

SEQUENCE 4：CGCTATCAAAACATCT。

Probe SEQUENCE1 is the probe at the 19th exon E746-A750del site wild-type, and probe SEQENCE2 is the probe at the 19th exon E746-A750del site mutation type.Primer SEQENCE 3, SEQENCE 4 are respectively specificity forward primer and the reverse primer at EGFR gene the 19th exon E746-A750del pleomorphism site.

The detection composition of the detection epidermal growth factor receptor gene polymorphism that the present invention relates to is the detection at a pleomorphism site of EGFR gene.In detection composition, probe can be common Taqman probe, also can be the MGB probe.Probe 5 ' end use the luminophore fluorescent mark, 3 ' end employing quenching group fluorescent mark.The mark fluorescent group of 5 ' end can be that intermediary such as FAM, ROX, TET, HEX, TAMRA, CY3, CY5, JOE, Texas red are a kind of, and the labelling groups of 3 ' end can be TAMRA, a kind of among the NFQ.

Simultaneously, the present invention also provides test kit of the present invention to determine this biological sample epidermal growth factor receptor gene the 19th exon polymorphism, the especially purposes of epidermal growth factor receptor gene the 19th exon E746-A750del loci polymorphism by the detection of biological sample.

Simultaneously, the present invention also provides the purposes of test kit of the present invention by the curative effect of detection of biological sample prediction epidermal growth factor receptor inhibitor treatment malignant tumour.

Wherein, if biological sample EGFR19 exon is an E746-A750del homozygous mutation type, then predict the good effect of epidermal growth factor receptor inhibitor treatment malignant tumour; If be heterozygous or wild-type, then predict the weak curative effect of epidermal growth factor receptor inhibitor treatment malignant tumour.

In purposes provided by the invention, described test kit comprises: polymerase chain reaction (PCR) amplification reagent composition, the detection composition, the epidermal growth factor receptor gene that detect the epidermal growth factor receptor gene polymorphism be wild/and mutator gene type positive control plasmid.

Wherein, the polymerase chain reaction (PCR) amplification reagent composition comprises Taq archaeal dna polymerase, polymerase chain reaction damping fluid, dNTP mixture, Mg ²⁺The ratio of a component is optimized through overtesting in the amplification.Wherein, the Taq archaeal dna polymerase, be can warm start the Taq archaeal dna polymerase.

Wherein, detect the detection composition of epidermal growth factor receptor gene polymorphism, comprising:

Probe:

SEQUENCE 1：VIC-CCCAGAAGGTGAGAAAGTTAAAATTC；

SEQUENCE 2：FAM-CCCACACAGC。

Primer:

SEQUENCE 3：ATTAAGAGAAGCAACATCT；

SEQUENCE 4：CGCTATCAAAACATCT。

Probe SEQUENCE1 is the probe at the 19th exon E746-A750del site wild-type, and probe SEQENCE 2 is the probe at the 19th exon E746-A750del site mutation type.Primer SEQENCE 3, SEQENCE 4 are respectively specificity forward primer and the reverse primer at EGFR gene the 19th exon E746-A750del pleomorphism site.

In purposes provided by the invention, EGF-R ELISA (EGFR) inhibitor medicaments is meant the inhibitor that is directed to EGFR butyric acid kinase activity zone, perhaps is called the tyrosine kinase inhibitor medicine.Preferably, the EGFR inhibitor of indication of the present invention is meant the small molecules tyrosine kinase inhibitor.Iressa Iressa) and erlotinib (English name: Erlotinib, trade(brand)name: Te Luokai Tarceva) more preferably, the small molecules tyrosine kinase inhibitor of indication of the present invention refers to Gefitinib (English name Gefitinib, trade(brand)name:.

In purposes provided by the invention, the detection of biological sample is DNA or mRNA sample, and preferably, the sample that the present invention is directed to is the cdna sample that derives from tumor tissues.In purposes provided by the invention, malignant tumour includes but not limited to lung cancer, colorectal cancer, mammary cancer, carcinoma of the pancreas, transitivity kidney and incidence squama cancer.

More preferably, the sample that the present invention is directed to is the sample that derives from cancerous lung tissue, especially derives from the genomic dna sample of nonsmall-cell lung cancer.

Employed gene-amplificative instrament comprises the quantitative real time PCR Instrument more than the 2 all passages in purposes provided by the invention, and the quantitative real time PCR Instrument of not establishing the grating control of passage.

The present invention is used to detect the genotypic process of sample:

1. detect the preparation of sample

As sample is dna sample, directly uses UV spectrophotometer measuring concentration of specimens and purity, and the OD260/OD280 ratio that requires sample is at 1.8-2.0, and sample is diluted to proper concentration, is specially between the 20ng/ml-400ng/ml.

As sample is mRNA, then needs reverse transcription reaction, carries out the preparation of sample then according to the requirement of DNA.

2. the amplification of sample target gene fragment

The dna sample that detects with desire is a template, uses and detects mixture and corresponding supporting positive control and negative control, on suitable gene-amplificative instrament, uses polymerase chain reaction (PCR) and carries out the gene amplification of corresponding pleomorphism site.On suitable gene-amplificative instrament, increase.

3. goal gene polymorphism fluorescent scanning and genotype are judged.

Amplified production is put into suitable real-time quantitative PCR instrument, carry out fluorescent scanning.The genotype of judging sample according to the scanning result and the fluorescent scanning result of positive control.

4. predict the epidermal growth factor receptor inhibitor curative effect

Carry out the outcome prediction of epidermal growth factor receptor inhibitor according to genotype.Concrete, if sample is exons 19 homozygous mutation types, predict that then curative effect of medication is good; If for heterozygous or wild-type then predict weak curative effect.

Advantage of the present invention is: with Auele Specific Primer ATTAAGAGAAGCAACATCT, CGCTATCAAAACATCT and probe VIC-CCCAGAAGGTGAGAAAGTTAAAATTC, FAM-CCCACACAGC is a core, at the reagent of preparation by pcr amplification biotinylated nucleic acid sample detection epidermal growth factor receptor gene the 19th exon polymorphism, and provide a kind of by detecting the test kit of pcr amplification biotinylated nucleic acid sample detection epidermal growth factor receptor gene the 19th exon polymorphism, test kit is determined this biological sample epidermal growth factor receptor gene the 19th exon polymorphism by the detection of biological sample, and the purposes for the treatment of the curative effect of malignant tumour by detection of biological sample prediction epidermal growth factor receptor inhibitor.Pcr template preparation method of the present invention is simple to operate, need not specific installation, thereby adopt the method that detects fluorescent signal directly to detect the PCR product behind the pcr amplification and obtain the genotype result, do not relate to as the electrophoretic pollution and the pollution that may cause for environment, and big flux high-level efficiency, the accuracy height, the large-scale crowd that is particularly suitable for clinical and scientific research uses, and is widely used.The 19th exon polymorphism result predicts the curative effect of epidermal growth factor receptor inhibitor according to epidermal growth factor receptor gene.The present invention has overcome the blindness of selection of clinical epidermal growth factor receptor inhibitor class medicine, can predict that on the basis of gene type assay the patient uses the validity and the security of epidermal growth factor receptor inhibitor class medicine, instruct clinical application, make the patient can carry out the individuation medical treatment, control tumour as early as possible effectively, reduce toxic side effect, reduce medical treatment cost.

The present invention has designed pcr amplification primer and probe at epidermal growth factor receptor gene the 19th exon pleomorphism site genotype especially, compare with the amplimer and the probe of routine, amplification efficiency height, specificity be good, save time, can directly identify the genotype in this site according to the clip size of PCR product, need not follow-up detection step and reagent corresponding, equipment, better use value is arranged.

Description of drawings

Fig. 1. plasmid clone spot pcr amplification is figure as a result.1.marker, the 2.PCR amplification.

Fig. 2 .40 name is taken the genotypic measurement result SDS data analysis of the nonsmall-cell lung cancer patient figure of Gefitinib.

Embodiment

Embodiment 1 epidermal growth factor receptor gene is wild/structure of mutator gene type positive control plasmid

Material and method

Instrument: whizzer (Eppendorf 5415D), PCR instrument (ABI 2700), gel imaging system (magnificent electric DH2000)

Material: pET28 vector plasmid, coli strain DH5 α

Operation steps:

1. tissue gene group DNA extraction:

Choose the tumor tissues of paraffin-embedded nonsmall-cell lung cancer, the dewaxing back uses DNA extraction test kit (QIAmp DNA kit) to extract genomic dna in the tumor tissues.

2.EGFR the segmental amplification of the 19th exon genes

Amplification EGF-R ELISA target gene fragment is used following primer amplification EGFR the 19th exon genes fragment.

SEQUENCE 5：CTCGAATTCCGTCTTCCTTCTCTCTCTGTC

SEQUENCE 6：TCCAAGCTTGACATGAGAAAAGGTGGGC

At 5 ' end of primer, include the restriction enzyme site of EcoR1 and Hind111 respectively

The PCR program:

95 ℃ of pre-sex change in 10 minutes, 95 ℃ 100 seconds, 60 ℃ 10 seconds, 72 ℃ 7 minutes, 45 circulations.72 ℃ were extended 5 minutes.

3. the clone of amplified fragments:

Enzyme is cut

Amplified fragments uses EcoR1 and HindIII double digestion, the enzyme tangent condition be 37 ℃ 1 hour, enzyme is cut product and is carried out purifying with purification kit.

Vector plasmid PET28 uses EcoR1 and HindIII double digestion equally, the enzyme tangent condition be 37 ℃ 1 hour, enzyme is cut product and is carried out purifying with purification kit.

Endonuclease bamhi connects

Adopt the T4 ligase enzyme, be connected according to 1: 2 ratio of carrier and amplified production, condition of contact is 16 ℃, 8 hours.

Transform

Connect product and transform DH5 α competence bacterium, transform the back, be coated with the flat board of Amp resistance then, be inverted and cultivated 10 hours 37 ℃ of shaking culture 2 hours.

Screening

With the clone's spot that grows on the flat board, adopt PCR method to clone evaluation, what qualification result coincide checks order.Therefrom filter out the correct clone of the wild-type of epidermal growth factor receptor gene and mutated genes type sequence as positive plasmid.

Pcr amplification the results are shown in Figure 1.

4. the amplification of positive plasmid and purifying.

Positive colony adopts the culturing bottle of 1L, a large amount of cultivation.Cultivate back alkali purification plasmid ,-20 ℃ of freezing preservations after spectrophotometer detection purity and the concentration.

Embodiment 2 utilizes test kit to carry out the genotypic mensuration of nonsmall-cell lung cancer sample

1. the preparation of sample

Get 40 parts of tumor tissues (flesh tissue or paraffin mass) of taking the nonsmall-cell lung cancer patient of Gefitinib, extract genomic dna.

2.PCR the configuration of amplification mixed solution

According to table 1 configuration pcr amplification mixed solution

Table 1PCR amplification mixed solution allocation list

Component	Volume ul
		Taq archaeal dna polymerase 5U/ul	0.5
10×PCR buffer	2.5
		Mg 2+25mM	3
dNTP 1.25mM	4
		Primer 1SEQUENCE3	0.4
Primer 2 SEQUENCE4	0.4
		Probe 1SEQUENCE1	0.25
Probe 2SEQUENCE2	0.25
		Dna profiling	11.25
H 2O	2.45
		Total	25

Dispose 40 parts of PCR reaction mixtures respectively, in addition, dispose two genotypic positive control mixed solutions and a negative sample mixed solution.

3.PCR amplified reaction

Reaction conditions is as follows: 95 ℃ 10 minutes, 92 ℃ 15 seconds, 60 ℃ of 40 circulations in 1 minute.

4. polymorphism analysis

The PCR reaction finishes the back and adopts the SDS data analysis software to carry out gene type assay, and analytical results is seen Fig. 2:

Can be learnt that by Fig. 2 VIC fluorescence is wild-type, FAM fluorescence is the homozygous mutation type, and middle fluorescence is heterozygous, can obtain table 2 according to collection of illustrative plates.

5. Gefitinib outcome prediction

According to the result of above-mentioned detection, genotype is after the patient of homozygous mutation type takes Gefitinib, and curative effect of medication is better than and takes Gefitinib minigene type is the patient of wild-type and heterozygous.

Table 240 is taken the nonsmall-cell lung cancer patient genotype of Gefitinib

＜110〉Hua'anfo Medicine Research Center Co., Ltd., Beijing

Shenzhen Aosa Medicine Co., Ltd

＜120〉test kit of prediction epidermal growth factor receptor inhibitor curative effect

<160>6

<170>PatentIn version 3.3

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Claims (14)

1. Auele Specific Primer that detects epidermal growth factor receptor gene the 19th exon E746-A750 del loci polymorphism with PCR method: ATTAAGAGAAGCAACATCT, CGCTATCAAAACATCT.

2. probe that detects epidermal growth factor receptor gene the 19th exon E746-A750 del loci polymorphism with PCR method: VIC-CCCAGAAGGTGAGAAAGTTAAAATTC, FAM-CCCACACAGC.

3. Auele Specific Primer ATTAAGAGAAGCAACATCT, CGCTATCAAAACATCT and probe VIC-CCCAGAAGGTGAGAAAGTTAAAATTC, FAM-CCCACACAGC are preparing by the purposes in the reagent of pcr amplification biotinylated nucleic acid sample detection epidermal growth factor receptor gene the 19th exon E746-A750 del loci polymorphism.

4. purposes according to claim 3 is characterized in that: contain Taq archaeal dna polymerase, dNTP mixture and Mg in the described reagent ²⁺

5. test kit that detects epidermal growth factor receptor gene the 19th exon E746-A750 del loci polymorphism comprises: polymerase chain reaction (PCR) amplification reagent composition, the detection composition that detects the epidermal growth factor receptor gene polymorphism and Urogastron be wild/and mutator gene type positive control plasmid.

6. test kit according to claim 5, wherein the polymerase chain reaction (PCR) amplification reagent composition comprises Taq archaeal dna polymerase, dNTP mixture, Mg ²⁺, the polymerase chain reaction damping fluid.

7. according to claims 6 described test kits, it is characterized in that: the detection composition that detects the epidermal growth factor receptor gene polymorphism comprises:

Probe: VIC-CCCAGAAGGTGAGAAAGTTAAAATTC, FAM-CCCACACAGC;

Auele Specific Primer: ATTAAGAGAAGCAACATCT, CGCTATCAAAACATCT.

8. according to the test kit described in the claim 7, it is characterized in that: probe is the MGB probe.

9. the test kit described in according to Claim 8 is characterized in that: 5 ' end luminophore fluorescent mark of probe, 3 ' holds and uses the quenching group fluorescent mark.

10. according to the test kit described in the claim 9, it is characterized in that: the mark fluorescent group of probe 5 ' end is selected from a kind of among FAM, ROX, TET, HEX, TAMRA, CY3, CY5, JOE, the Texas red, and the labelling groups of 3 ' end is TAMRA or NFQ.

11. test kit according to claim 5 detects the purposes of epidermal growth factor receptor gene the 19th exon polymorphism.

12. purposes according to claim 11 is characterized in that detection site is epidermal growth factor receptor gene the 19th exon E746-A750 del site.

13. test kit according to claim 5 is by the purposes of the curative effect of detection of biological sample prediction epidermal growth factor receptor inhibitor treatment malignant tumour.

14. purposes according to claim 13 is characterized in that: malignant tumour comprises lung cancer, colorectal cancer, mammary cancer, carcinoma of the pancreas, transitivity kidney and incidence squama cancer.

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