CN1749417A - Real time quantitative PCR detecting reagent kit and method for epidermal growth factor receptor exon 19 deletion mutation - Google Patents

Abstract

The present invention relates to one method of real time quantitative PCR detecting exon 19 deletion mutation of epidermal growth factor receptor in sample, and the method includes: A. real time quantitative PCR detection of sample with the first reagent set to obtain the gene copy number of the wild epidermal growth factor receptor exon 19; B. detection the sample with the second reagent set to obtain the total copy number of amplified product in the sample; and judging whether to have deletion mutation in the epidermal growth factor receptor exon 19 based on the ratio of the copy numbers obtained in the steps A and B.

Description

The real time quantitative PCR detecting reagent kit and the method for epidermal growth factor receptor exon 19 deletion mutations

Technical field

The present invention relates to the medical science oncology.Specifically, the invention describes novel method and the test kit thereof that a kind of real-time quantitative PCR detects the variation of epidermal growth factor receptor exon 19 genetically deficients.

Background technology

In recent years, with epithelial growth factor receptor (Epidermal Growth Factor Receptor, EGFR) the molecular targeted treatment (Molecular Targeted Therapy) for the treatment target has been subjected to the common concern of domestic and international tumour circle, wherein EGFR tyrosine kinase inhibitor Genfitinib (claim not only Iressa) and Erlotinib (but also claiming Tarceva) are used for the treatment of nonsmall-cell lung cancer in late period (NSCLC) by FDA (Food and Drug Adminstration) (FDA) approval, and Gefitinib also obtains to comprise Japan respectively, the approval of 27 countries such as Australia, China has also ratified Genfitinib in February, 2005 and has entered clinical.

Data shows that to going through repeatedly the advanced NSCLC of chemotherapy failure, Genfitinib and Erlotinib pharmacological agent are first-selected at present treatment measures, can make patient's remission, and focus is dwindled, and quality of life improves, and prolong lifetime.The clinical trial of these other noumenal tumours of class pharmacological agent (as mammary cancer and large bowel cancer) is also in process.But nearest discovers, the patient overwhelming majority of Genfitinib and Erlotinib treatment produce effects is EGFR transgenation person, and this class patient accounts for the 10%-30% of NSCLC sum, and invalid substantially to wild-type tumour said medicine.Thereby EGFR gene in the patient tumors both detected and can select treatment target targetedly, the patient is benefited to greatest extent, the patient medical expense that can avoid non-rational use of drug to cause again increases and social medical resource is wasted.This gene diagnostic new technology is applied to clinical, and millions of cancer patients are benefited, and its social effect and economic worth are all very remarkable.

People EGFR gene is positioned at the short arm of a chromosome 7p12-14 district No. 7, is made up of 28 exons.But transgenation occurs in exons 1 9-21 more than 90%, and these sudden changes can be divided into three types:

A) base deletion of exons 19.It mainly is the base deletion sudden change of 746-752 bit codon, cause amino acid in the EGFR albumen (ELREATS) sequence to be lost, this disappearance has changed acceptor ATP in conjunction with capsule (ATP-binding pocket, abbreviation ABP) angle, thus the susceptibility of cancer cells significantly strengthened to Gefitinib and Erlotinib.

B) point mutation of extron 20 or base are inserted sudden change.Point mutation mainly is that C＞T conversion appears in the 790th bit codon, the amino acid that causes this site in the EGFR albumen changes methionine(Met) (being called for short T790M) into by Threonine, this sudden change is detected in recidivist after the pharmacological agent, and sudden change makes cancer cells produce resistance to Gefitinib and Erlotinib.Base is inserted and is mutated present 770-775 bit codon, and there are 8 kinds of different inserted modes in (mainly in ccccc sequence both sides) between gac aac ccc cac gtg tgc sequence, and the fragment of insertion is a 3-9 base.The clinical meaning that this class is inserted sudden change it be not immediately clear.

C) point mutation of exon 21.Mainly be that 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.

American scholar Shigematsu etc. have carried out largest in the world so far clinical study, and they have analyzed 617 routine cancerous lung tissues, find that the incidence of exons 19 transgenations is the highest, account for more than 50% of EGFR transgenation sum.In addition, the current research of Japanese scholar Mitsudomi etc. finds that the dependency of exon 19 deletion mutantions and Gefitinib clinical efficacy is best in all EGFR transgenations.And reports such as Korea S scholar Lee, about 7% deletion mutantion that has an EGFR exons 19 points out this sudden change to be not limited only to lung cancer in the incidence squama cancer, and also there is similar sudden change in other malignant entity tumors.Therefore, the gene test of exons 19 sudden changes has been subjected to great attention.

According to incompletely statistics, exons 19 transgenations of having found both at home and abroad at present have 22 kinds of different types (table 1) at least, but the gene mutation spectrum of exon 19 is not illustrated at present as yet fully in Chinese's lung cancer.From available data the result of study of totally 218 routine tumours (comprising 2 pieces of documents of continent scholar totally 117 example and 1 piece of document 101 example of Taiwan's scholars) add up, the incidence of EGFR exons 1 9-21 transgenation is about 34.4% (75/218 example) in China's lung cancer, wherein exons 19 sudden changes account for 48% (36/75 example), the extron 20 sudden change accounts for 6.7% (5/75 example), and exon 21 sudden change accounts for 45.3% (34/75 example).In addition, there is regional disparity in the EGFR transgenation in Chinese's lung cancer, reports that as the continent scholar mutation rate (19.7%, 23/117 example) of exons 19 is higher than exon 21 (10.3%, and Taiwan results reported opposite (12.9%vs.21.8%) 12/117 example).But two places the scholar all report, the mutation rate of extron 20 is very low, is respectively 0.9% (continent) and 4.0% (Taiwan).

Up to now, found that exon 19 transgenations have 9 types (table 2) in Chinese's lung cancer, but main type is that delE746-A750[comprises del E746-A750 (1) and del E746-A750 (2)], account for 61% (22/36 example).

The gene mutation type of table 1 EGFR exons 19

Numbering	Mutation type	Base sequence (5 '-3 ')
				Wild-type	g c t a t c a a g g a a t t a a g a g a a g c a a c a t c t c c g a a a
1	del E746-A750(1)	g c t a t c a a - - - - - - - - - - - - - - - - - - a a c a t c t c c g a a a
			2	del E746-A750(2)	g c t a t c a a g - - - - - - - - - - - - - - - - - - a c a t c t c c g a a a
3	del E746-A750ins T	g c t a t c a - - - - - - - - - - - - - - - - - - c a a c a t c t c c g a a a
			4	del E746-A750ins P	g c t a t c a a g - - - - - - - - - - - - - - - - - - c c a t c t c c g a a a
5	del E746-A750ins I	g c - - - - - - - - - a a t t a a g a t - - - - - - - - - a t c t c c g a a a

6	del E746-T751	g c t a t c a a - - - - - - - - - - - - - - - - - - - - - - a t c t c c g a a a
			7	del E746-T751ins I	g c t a t c a a - - - - - - - - - - - - - - - - - - - - - - a a t t c c g a a a
8	del L747-E749	g c t a t c a a g g a a - - - - - - - - - - - g c a a c a t c t c c g a a a
			9	del L747-E749insP	g c t a t c a a g g a a - - - - - - - - - - - c c a a c a t c t c c g a a a
10	del L747-A750insP	g c t a t c a a g g a a - - - - - - - - - - - - - - - c c a t c t c c g a a a
			11	del L747-T751	g c t a t c a a g g a a t - - - - - - - - - - - - - - - - - - c t c c g a a a
12	del L747-T751insP	g c t a t c a a g g a a c - - - - - - - - - - - - - - - - - - c t c c g a a a
			13	del L747-T751ins V	g c t a t c a a g g - - - - - - - - - - - - - - - - - - t a t c t c c g a a a
14	del L747-T751ins A	g c t a t c a a g g - - - - - - - - - - - - - - - - - - c a t c t c c g a a a
			15	de l L747-S752	g c t a t c a a g g a a - - - - - - - - - - - - - - - - - - - - - c c g a a a
16	del L747-S752ins V(1)	g c t a t c a a g g - - - - - - - - - - - - - - - - - - - - - t t c c g a a a
			17	del L747-S752ins V(2)	g c t a t c a a g g - - t t - - - - - - - - - - - - - - - - - - - - c c g a a a
18	del L747-S752ins A	g c t a t c a a g g - - - - - - - - - - - - - - - - - - - - - c t c c g a a a
			19	del L747-S752ins D	g c t a t c a a g g a - - - - - - - - - - - - - - - - - - - - - t c c g a a a
20	del L747-S752ins Q(1)	g c t a t c a a g g a a - - - - - - - - - - - - c a a - - - - - - c c g a a a
			21	del L747-S752ins Q(2)	g c t a t c a a g g a a - - - - - - - - - - - - - - - - - - - - - c a g a a a
22	del L747-S752ins S	g c t a t c a a g g a a t - - - - - - - - - - - - - - - - - - - - - c g a a a

The main type of table 2 Chinese lung cancer EGFR exons 19 transgenations

Numbering	Mutation type	Base sequence (5 '-3 ')
				Wild-type	g c t a t c a a g g a a t t a a g a g a a g c a a c a t c t c c g a a a
1	del E746-A750(1)	g c t a t c a a - - - - - - - - - - - - - - - - - - a a c a t c t c c g a a a
			2	del E746-A750(2)	g c t a t c a a g - - - - - - - - - - - - - - - - - a c a t c t c c g a a a

9	del L747-E749insP	g c t a t c a a g g a a - - - - - - - - - - - c c a a c a t c t c c g a a a
			10	del L747-A750insP	g c t a t c a a g g a a - - - - - - - - - - - - - - - c c a t c t c c g a a a
11	del L747-T751	g c t a t c a a g g a a t - - - - - - - - - - - - - - - - - - c t c c g a a a
			14	del L747-T751ins A	g c t a t c a a g g - - - - - - - - - - - - - - - - - - c a t c t c c g a a a
19	del L747-S752ins D	g c t a t c a a g g a - - - - - - - - - - - - - - - - - - - - - t c c g a a a
			21	del L747-S752ins Q(2)	g c t a t c a a g g a a - - - - - - - - - - - - - - - - - - - - - c a g a a a
22	del L747-S752ins S	g c t a t c a a g g a a t - - - - - - - - - - - - - - - - - - - - - c g a a a

The standard detecting method of exons 19 transgenations at present is direct order-checkings of tissue DNA sample, but there are some weak points in this gene tester:

The one, the DNA requirement is big, the collection difficulty of detection sample.It is generally acknowledged, tissue DNA directly carries out gene sequencing behind pcr amplification, the DNA requirement is at least more than 100ng, and the unique approach that can obtain to organize of patients with advanced cancer is aspiration biopsy, but the sample of tumour aspiration biopsy tissue is very little, usually only contain tens thousand of cells, thereby the EGFR gene test of microcomponent's sample is quite difficult, at present the domestic report that does not also have success.

The 2nd, this method is to the requirement height of technology, interpretation time and effort consuming as a result.Though gene sequencing is the most intuitive and accurate detection method, scope and the type thereof that can clarify a diagnosis and suddenly change, but whole testing process needs just can finish 3-5 working days, and, accurately diagnose the mutation type difficulty bigger because the great majority sudden change belongs to heterozygosity sudden change (promptly having wild-type and mutant amplified production simultaneously).Thereby this method only is fit to the superior laboratory of minority technical equipment and carries out, and the domestic hospital that can carry out this diagnosis is very few.

Therefore, the detection of EGFR transgenation will become a routine clinical diagnostic techniques, presses for highly sensitive, accurate, quick, the easy novel method of research and development.

Real-time quantitative PCR (Real-time Quantitative PCR) is a novel technique of gene detection that grows up nineteen nineties.This technology has the following advantages:

1. highly sensitive.Because real-time quantitative PCR adopts detection technique of fluorescence, thereby susceptibility significantly increases than other gene testers, it is generally acknowledged that its minimum limit of surveying reaches 0.1-1pg DNA.

2. specificity is good.Real-time quantitative PCR detect to require 200bp with interior target gene dna fragmentation on one group of primer of design and fluorescent probe, thereby the base sequence more than 3/4 must could obtain fluorescent signal with the primer probe is complementary in this fragment, and this has just guaranteed that detected result has good specificity.

3. easy, quick, high-throughput.The real-time quantitative PCR technology is handled by the automatization of instrument, can finish the analysis of 30-100 example sample simultaneously and directly provide the result about 1 hour, thereby data analysis is intuitively easy.Owing to saved the operations behind the pcr amplification, both save the time, alleviated labour intensity simultaneously, reduced the chance that causes DNA to pollute because of these operations again.

Japan scholar Sasaki etc. at first adopts the real-time quantitative PCR technology to carry out the gene test research of EGFR, and they adopt the hybridization probe method that exon 19 is carried out gene type, have obtained satisfactory result.But also there are some shortcomings in this method, because in known 22 kinds of transgenations at present, the author only carried out analysis to wherein 2 types (being del E746-A750 (1) and delE746-A750 (2)), can this method diagnose other mutation types of exons 19, and still lacking data at present can be for judging.

Summary of the invention

At the problems referred to above, the present inventor has at first carried out large-scale clinical study, and 120 routine lung cancer have been carried out the gene sequencing of exons 19, detects 6 kinds of mutation types altogether.According to the gene mutation spectrum of exons 19, the present inventor has researched and developed a kind of new real-time quantitative PCR detection technique, can detect all 22 kinds of exons 19 transgenations.

For achieving the above object, first aspect present invention provides a kind of test kit by real-time quantitative PCR detection epidermal growth factor receptor exon 19 deletion mutations, it is characterized in that described test kit is made up of following:

(a) epithelial growth factor receptor alia gene standard substance;

(b) first reagent set, this reagent set contains a pair of primer and Taqman probe, described primer is to being that ATP to epidermal growth factor receptor exon 19 codings has specific primer in conjunction with the capsule sequence, described Taqman probe has corresponding to 19 the 746th nucleotide sequences to the encoding sequence of 752 bit codons of wild-type epidermal growth factor receptor exon, its 5 ' end links to each other with the fluorescence report group, and 3 ' end links to each other with the fluorescent quenching group;

(c) second reagent set, this reagent set comprise a pair of primer and DNA dyestuff, and the primer in wherein said primer pair and first reagent set is to identical.

Particularly, test kit of the present invention is made up of EGFR gene standard substance and two reagent set.First reagent set (being called for short ABP reagent) has comprised a pair of primer and a Taqman probe, be characterized in, described primer all has binding site to wild-type and mutant egf R albumin A TP in conjunction with the encoding sequence (EGFR gene extron 19) of capsule (ATP Binding Pocket is called for short ABP).In a preferable embodiment, described primer has the sequence shown in SEQ ID NO:5 and the SEQ ID NO:6.And the Taqman probe has the nucleotide sequence corresponding to exons 19 746-752 bit codon encoding sequences, as has the nucleotide sequence shown in the SEQ ID NO:7.Therefore, using ABP reagent carries out the PCR reaction to wild-type and mutant exons 19 and all can obtain amplified production, wherein the wild type gene product is owing to have the complete binding sequence of Taqman probe, thereby produced fluorescent signal at the reaction process middle probe by the Taq enzymic hydrolysis, and strength of signal is directly proportional with template content.And the mutated genes product only has the part binding sequence of probe, thereby probe can be had fluorescent signal (reaction principle of ABP detection reagent as shown in Figure 2) so have only amplified production in the PCR reaction by the Taq enzymic hydrolysis in reaction process.

Second reagent set (being called SYB reagent) in the test kit of the present invention comprises a pair of primer and DNA dyestuff, and the primer in wherein said primer pair and first reagent set is to identical.Described DNA dyestuff is preferably the SYBR Premix Ex Taq of Syb Green I[such as the production of precious biotechnology (Dalian) company limited ^TMProduct, catalog number (Cat.No.) DRR041D].Because in above-mentioned reaction system, wild-type and mutant exons 19 all can be amplified, thereby adopt DNA dyestuff Syb Green I to measure to the total amount of amplified production.

In addition, this test kit provides the known EGFR gene expression plasmid of copy number as standard substance, after using this standard substance preparation standard curve, respectively sample to be tested DNA is detected with ABP reagent and SYB reagent, can carry out digitized processing to the result, obtain the gene copy number (being called ABP) of wild-type exons 19 and total copy number (being called SYB) of amplified production,, just can draw detected result by calculating ABP/SYB ratio.

Therefore, the invention provides a kind of method by epidermal growth factor receptor exon 19 deletion mutations in the real-time quantitative PCR test sample, this method comprises, (a) with first reagent set of the present invention sample is carried out real-time quantitative PCR and detect, obtain the gene copy number of wild-type epidermal growth factor receptor exon 19 in the sample; (b) with second reagent set of the present invention sample is detected, obtain total copy number of amplified production in the sample; Whether there are epidermal growth factor receptor exon 19 deletion mutations according to step (a) with in the ratio in judgement sample of the copy number that (b) obtains.

A remarkable advantage of test kit of the present invention has the specificity of height.Experimental result proves, at present known all mutated genes is used present method can both obtain diagnostic result.Another advantage of the present invention has the detection sensitivity of height, experimental result proves, mutated genes is lower than 10% in the sample DNA, the DNA total amount is lower than 2.5ng, use present method and can both obtain diagnostic result, thereby present method is more suitable for, and basic medical unit is conventional to be used.

The accompanying drawing summary

Fig. 1 has shown that there are 5 in the base insertion sudden change of EGFR extron 20 and insert sites (two line part) in extron 20, and it inserts fragment 8 kinds of different sequences.

Fig. 2 has shown the quantitative PCR detection synoptic diagram of EGFR exons 19 deletion mutantions.

Fig. 3 has shown the sequencing result of 6 kinds of gene mutation types of EGFR exons 19.

Fig. 4 has shown the genetically deficient sudden change of cancerous lung tissue EGFR exons 19.

Fig. 5 has shown the result of quantitative PCR standard substance preparations, wherein the PCR product before the 1 expression purifying; PCR product behind the 2 expression purifying; 2 positive expression plasmid electrophoresis result that 3,4 expressions obtain through screening.

Fig. 6 A has shown the typical curve (RG3000 type quantitative PCR instrument result) that the real-time quantitative PCR of ABP detects.

Fig. 6 B has shown the typical curve (RG3000 type quantitative PCR instrument result) that the real-time quantitative PCR of SYB detects.

Fig. 7 has shown the specificity analyses of ABP detection reagent.Wild-type and mutant exons 19 are carried out quantitative PCR detection (A), get amplified production after reaction finishes and carry out 1.6% agarose gel electrophoresis 100V 30min, sheet (B) is taken the photograph in EB dyeing.Wherein swimming lane 1-6 is the wild-type dna profiling, and 7-12 is the mutant DNA template, wherein 1,7: blank; 2,8:10,000pg; 3,9:1,000pg; 4,10:100pg; 5,11:10pg; 6,12:1pg.

Preferable embodiment

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Unless description is arranged in addition, enforcement of the present invention will be adopted the routine techniques of molecular biology, recombinant DNA, and these all are known to those skilled in the art.

Embodiment 1: the gene sequencing of cancerous lung tissue EGFR exons 19

(1) materials and methods

A. tumor tissues

Gather cancerous lung tissue 120 examples of excision, types of organization is adenocarcinoma of lung 48 examples, adenosquamous carcinoma 24 examples, lung squamous cancer 41 examples, other types 7 examples.The male sex's 85 examples wherein, women's 35 examples, the age is 37-79 year, average out to 60.4 years old.Tumor tissues quick freezing in back 1 hour of collection is stored in the liquid nitrogen, and ℃ profound hypothermia refrigerator is preserved then-80.

B.DNA extracts

Get the 50mg tissue samples, adopt DNAezsol genome DNA extraction test kit (company can be widely collected in the Shen, Shanghai) to extract genomic dna, the DNA extraction process is operated according to the test kit specification sheets.

Pcr amplification and gene sequencing

Publish people's epithelial growth factor receptor (EGFR) gene (AY588246) sequence according to the Genbank database, the Primer Express software that adopts u.s.a. applied biosystem (ABI) company is respectively at exon 19 design following primers (table 3).Above-mentioned primer is synthetic by match Parkson, Shanghai gene engineering company limited.

The PCR primer of table 3 EGFR gene extron 19

Exon	Primer	Nucleotide position	Base sequence (5 '-3 ')	Tm	Product
						19	The downstream, upstream	157524-157547 157653-157673	aac gtc ttc ctt ctc tct ctg tca(SEQ ID NO：1) cca cac agc aaa gca gaa act (SEQ ID NO：2)	57 57	150 bp

The pcr amplification of DNA adopts Pfu high-fidelity DNA polymerase (available from Lithuania MBI Fermantas company), the PCR reaction solution includes DNA 4 μ l (100ng), 10 * damping fluid, 5 μ l, 2.5mM dNTP 5 μ l, each 1 μ l of upstream and downstream primer (20 μ M), Pfu enzyme (2.5U/ μ l) 1 μ l uses DEPC-H ₂O is supplemented to 50 μ l.The PCR reaction conditions is 95 ℃ of sex change in 5 minutes, then 95 ℃ 30 seconds, 57 ℃ 30 seconds, 72 ℃ 45 seconds, 40 circulations; 72 ℃ were extended 7 minutes.

The PCR product is used for 1.6% agarose gel electrophoresis, and condition is 100V 30 minutes, and gel is taken the photograph sheet after EB dyeing.The PCR product adopts upstream primer to carry out gene sequencing behind column separating purification, and examining order is finished by Shanghai branch office of Beijing AudioCodes Bioisystech Co., Ltd.

(2) result

EGFR exons 19 genes in the China mainland 120 routine cancerous lung tissues are detected, detect sudden change 16 examples altogether, the recall rate of exons 19 transgenations is 13.3%.These 16 routine exon 19 transgenations are heterozygosity sudden change (promptly having wild-type and mutated genes simultaneously), are divided into 6 types (table 4):

Class1 (693) has lacked 15 bases, causes EGFR albumen 746-750 amino acids (E L R E A) to be lost, and is called for short del E746-A750.

The base deletion site of type 2 (864) is moved 1 after than Class1, also lacks 15 bases, but that the amino acid that disappearance causes is lost is identical with Class1, thereby this type sudden change also can classify as del E746-A750.

Type 3 (775) has lacked 9 bases, and (t t a a g a g a a), and guanine thereafter (G) is converted to cytosine(Cyt) (C), cause EGFR albumen 747-749 amino acids (L R E) to be lost, introduce 1 new sudden change simultaneously, become proline(Pro) (P) by original L-Ala (A), be called for short del L747-E749insP.

The disappearance number of type 4 (201) increases by 4 bases than type 3, and VITAMIN B4 thereafter (A) is converted to cytosine(Cyt) (C), cause EGFR albumen 747-750 amino acids (L R E A) to be lost, introduce 1 new sudden change simultaneously, become proline(Pro) (P) by original L-Ala (A), be called for short del L747-A750insP.

Type 5 (251) has lacked 18 bases, causes EGFR albumen 747-752 amino acids (L R E A T S) to be lost, and is called for short del L747-S752.

Type 6 (694) is made up of two base deletions, cause EGFR albumen 746-752 amino acids (L R E A T S) to be lost, introduce 1 new sudden change simultaneously, become a word used in person's names propylhomoserin (V), be called for short delE746-S752insV by 746 original glutamine (E).

The sequential analysis collection of illustrative plates of said mutation is seen Fig. 3.

6 kinds of gene mutation types of table 4 EGFR exons 19

Numbering	Base sequence (2416-2448)	Aminoacid sequence (744-754)
			Wild type 693 864 775 201 251 694	a t c a a g g a a t t a a g a g a a g c a a c a t c t c c g a a a a t c a a - - - - - - - - - - - - - - - - - - a a c a t c t c c g a a a a t c a a g - - - - - - - - - - - - - - - - - - a c a t c t c c g a a a a t c a a g g a a - - - - - - - - - - c c a a c a t c t c c g a a a a t c a a g g a a - - - - - - - - - - - - - - - c c a t c t c c g a a a a t c a a g g a a - - - - - - - - - - - - - - - - - - - - - - c c g a a a a t c a a g g - - t t - - - - - - - - - - - - - - - - - - - - c c g a a a	I K E L R E A T S P K I K - - - - - - - T S P K I K - - - - - - - T S P K I K E - - - - - P T S P K I K E - - - - - - P S P K I K E - - - - - - - - P K I K V - - - - - - - - P K

Compare with table 4 result, have 2 kinds [Del L747-S752 and Del L747-S752ins V (2)] to find first in above-mentioned 6 types of sudden changes, all the other the 4 kinds existing reports of sudden change.

Table 5 result demonstration, from sex and types of organization, women's (7/35 example among the exons 19 transgenation persons, 20%) is higher than male sex's (9/85 example, 10.6%), gland cancer (13/48 example, 27.1%) is higher than adenosquamous carcinoma (1/24 example, 4.2%) and squama cancer (2/41 example, 4.9%).And from the mutation type analysis, 2 types Del E746-A750 sudden change accounts for 2/3 (11/16 example, 68.8%), and other 4 types account for 1/3 (5/16 example, 31.3%).The above results and domestic literature report basically identical.

The clinical related data of table 5 EGFR exons 19 transgenations

Numbering	Sex	Age	Types of organization	Clinical stages	Mutation type
						59	The man	74	The squama cancer	T4N0M1	Del E746-A750(2)
60	The woman	70	Gland cancer	T4N1M1	Del E746-A750(1)
						61	The man	54	Adenosquamous carcinoma	T4N2M1	Del E746-A750(2)
63	The man	72	The squama cancer	T4N2M0	Del E746-A750(2)
						201	The woman	65	Gland cancer	T2N1M0	Del L747-A750insP
229	The man	40	Gland cancer	T4N2M0	Del E746-A750(1)
						231	The man	65	Gland cancer	T3N2M0	Del E746-A750(1)
228	The man	63	Gland cancer	T2N0M0	Del E746-A750(1)
						240	The woman	48	Gland cancer	T2N2M0	Del E746-A750(1)
251	The man	50	Gland cancer	T3N2M0	De l L747-S752
						655	The woman	66	Gland cancer	T2N0M0	Del E746-A750(1)
693	The woman	54	Gland cancer	T4N2M0	Del E746-A750(1)
						694	The man	58	Gland cancer	T2N2M0	Del L747-S752ins V(2)
775	The woman	56	Gland cancer	T2N0M0	Del L747-E749ins P
						862	The man	44	Gland cancer	T2N3M0	Del L747-E749ins P
864	The woman	62	Gland cancer	T2N2M0	Del E746-A750(2)

Embodiment 2: EGFR exons 19 gene clones of sudden change tissue

(1) materials and methods

Tissue DNA adopts the Taq enzyme to carry out pcr amplification, the PCR reaction solution includes 10 * damping fluid, 5 μ l, 2.5mM dNTP5 μ l, each 1 μ l of EGFR exons 19 upstreams and downstream primer (20 μ M), the Taq enzyme is (available from precious biotechnology Dalian company limited, 2.5U/ 1 μ l μ l), tissue DNA 4 μ l (100ng) use DEPC-H ₂O is supplemented to 50 μ l.

The PCR reaction conditions is 95 ℃ of 5min sex change, 95 ℃ of 45sec. then, 56 ℃ of 2min, 40 circulations; 72 ℃ are extended 10min.The PCR product cuts behind 1.6% agarose gel electrophoresis 100V 30min and obtains amplified production, adopts then amplified production is carried out purifying.

Purified product is cloned in the pMD 18-T carrier (available from precious biotechnology Dalian company limited).Obtain the positive expression plasmid according to the operation instructions screening.

The gene sequencing of plasmid purification is finished by Shanghai branch office of Beijing AudioCodes biotech company.

(2) result

Gene clone has been carried out in 6 types of exons 19 deletion mutantions among Fig. 3, and screening obtains clone's 120 strains altogether, and sequencing result shows wild type gene 105 strains, mutated genes 15 strains, the sequence of mutated genes and table 4 described (Fig. 4) in full accord.

Embodiment 3: the preparation of real-time quantitative PCR standard substance plasmid

(1) materials and methods

EGFR positive expression cell: people A431 epithelium sample JEG-3 is biochemical and RESEARCH ON CELL-BIOLOGY institute available from Chinese Academy of Sciences Shanghai.Foetal calf serum and F12 substratum are available from GIBCO company.Tri Reagent RNA extraction agent box is available from U.S. Molecular Research Center company.RevertAid ^TMThe first chain cDNA synthetic agent box is available from Lithuania MBIFermantas company.PCR primer and fluorescent probe are synthetic by match Parkson, Shanghai gene engineering company limited.The order-checking of PCR product is finished by Shanghai branch office of Beijing AudioCodes biotech company.Ex TaqDNA polysaccharase and pMD 18-T PCR product cloning carrier and real-time quantitative PCR test kit are available from precious biotechnology (Dalian) company limited.

RNA extracting and cDNA are synthetic: select to enliven propagation phase cell, by test kit requirement extracted total RNA, adopt determined by ultraviolet spectrophotometry A260 and A280, require A260/A280＞1.80.Get 2 μ g RNA then by the synthetic cDNA of test kit requirement, reaction volume is 20 μ l.

Design of primers:

Publish people's epithelial growth factor receptor (EGFR) cDNA (X00588) sequence according to the Genbank database, adopt u.s.a. applied biosystem (ABI) company Prism Express software design 1 group of primer (table 6) (EGFR gene (AY588246) the 28th exon sequence is the sequence of EGFR gene, comprises intron and exon; And epithelial growth factor receptor (EGFR) cDNA (X00588) sequence is the cDNA sequence of EGFR, and the exon that is equivalent to the EGFR gene joins end to end in order, does not contain intron).

Table 6 PCR standard substance primer sequence

Primer	Nucleotide position	Base sequence (5 '-3 ')	Tm (C)	Product (bp)
					Upstream 1	2358	cac ggt gta taa ggg act ctg(SEQ ID NO：3)	57	1372
Downstream 1	3729	ggc ttc ctt ggg aaa gaa gt(SEQ ID NO：4)	56

The pcr amplification of cDNA: use the plasmid standard primer cDNA is carried out pcr amplification.The PCR reaction solution includes cDNA1 μ l, 10 * Ex Taq damping fluid, 5 μ l, and 2.5mM dNTP 5 μ l, each 1 μ l of upstream and downstream primer (20 μ M), Ex Taq (5U/ μ L) 0.25 μ l uses DEPC-H ₂O is supplemented to 50 μ l.The PCR reaction conditions is 95 ℃ of 5min sex change, 95 ℃ of 45sec. then, 56 ℃ of 2min, 40 circulations; 72 ℃ are extended 10min.Sheet is taken the photograph in amplified production EB dyeing behind 1.6% agarose gel electrophoresis 100V 30min.

Gene clone and standard substance screening method are as mentioned above.

(2) result

The A431 cell is the strain of present internationally recognized EGFR high expressing cell.Get this cell preparation cDNA, obtained single band through pcr amplification, this product is (Fig. 5 A) behind the gel separation purifying, obtains positive expression plasmid (Fig. 5 B) by clone and antibiotic screening, and the latter confirms that through gene sequencing its sequence is a wild-type.This plasmid is as the standard substance of quantitative PCR detection.

Embodiment 4: the foundation of real-time quantitative PCR detection method

(1) materials and methods

A. primer probe design

Publish people's epithelial growth factor receptor (EGFR) gene (AY588246) exons 19 sequences according to the Genbank database, adopt the Prism Express software of u.s.a. applied biosystem (ABI) company, designed 1 group of primer and Taqman probe (table 7) respectively.

Table 7 EGFR gene extron 19 and 28 primer probe sequence

Primer	Nucleotide position	Sequence (5 '-3 ')	Tm (℃)	Product (bp)
					The upstream	157553	act ctg gat ccc aga agg tga g(SEQ ID NO：5)	59	91
The downstream	157643	gat ttc ctt gtt ggc ttt cgg(SEQ ID NO：6)	60
				The Taqman probe	157586	FAM-tgc Ttc tct taa ttc ctt gat agc gac ggg a-TEMRA(SEQ ID NO：7)	70

B. reagent preparation

Plasmid standard: plasmid standard is diluted to 5 * 10 ⁹Be distributed into 10 μ l/ behind the copy/μ l and prop up ,-70 ℃ of refrigerators are preserved.The volumetric molar concentration of standard substance gene copy number=plasmid * 6.023 * 10 ²³

ABP reagent: reaction solution includes 5x PCR in real time damping fluid 5 μ l[test kits available from precious biotechnology (Dalian) company limited, Takara Ex Taq ^TMR-PCR Version 2.1, catalog number (Cat.No.) DRR032A), 10mM dNTP 0.75 μ l, 250mM Mg solution 0.5 μ l, warm start Taq enzyme (5U/ml) 0.25 μ l, each 0.1 μ l of upstream and downstream primer (100 μ M), fluorescent probe (50 μ M) is used DEPC-H ₂O is supplemented to 23 μ l.Be distributed into 950 μ l/ after the reaction solution preparation and prop up ,-70 ℃ of refrigerators are preserved.

SYB reagent: reaction solution includes 2 * SYBR Primix Ex Taq mixed solution, 12.5 μ l[test kits available from precious biotechnology (Dalian) company limited, SYBR Premix Ex Taq ^TM, catalog number (Cat.No.) DRR041D], each 0.1 μ l of upstream and downstream primer (100 μ M), 50 * ROX internal reference dyestuff (reagent source is the same), 0.5 μ l uses DEPC-H ₂O is supplemented to 23 μ l.Be distributed into 950 μ l/ after the reaction solution preparation and prop up ,-70 ℃ of refrigerators are preserved.

C. the preparation of typical curve and detection

1. take out standard QC and reaction mixture pipe, place 4 ℃ of refrigerators and treat ice-out.

2. the dilution of standard substance:

(1) gets 4 of 0.5ml Eppendorf centrifuge tubes, respectively add DEPC-H ₂O 18 μ l;

(2) the standard QC is of short duration centrifugal behind the mixing gently, get 2 μ l standard substance in No. 1 pipe, mixing 10 times;

(3) in No. 1 pipe, get 2 μ l standard substance, place in No. 2 pipes mixing 10 times;

(4) in No. 2 pipes, get 2 μ l standard substance, place in No. 3 pipes mixing 10 times;

(5) in No. 3 pipes, get 2 μ l standard substance, place in No. 4 pipes mixing 10 times;

3. in 0.2ml PCR thin-walled tube (or PCR kapillary), add 23 μ l reaction mixtures.

4. in blank pipe (No. 0 pipe), add 2 μ l DEPC-H ₂O adds the standard substance 2 μ l that manage dilution for 0-5 number successively by above-mentioned order in the standard QC, of short duration centrifugal behind the mixing gently, each is managed contained standard substance copy number and is respectively:

The pipe copy number

0 0

1 1×10 ⁸

2 1×10 ⁷

3 1×10 ⁶

4 1×10 ⁵

5. be sample hose after the standard pipe series, add 2 μ l sample DNAs successively, of short duration centrifugal behind the mixing gently.

6. then standard QC and sample hose are put into the PCR instrument successively.

7.PCR response procedures:

Insulation	Sex change		45 circulations	Insulation
						50 ℃ 300 seconds	95 ℃ 30 seconds	(1) 95 ℃ 20 seconds (2) 60 ℃ 60 seconds	37 ℃ 30 seconds

On RG300 type quantitative PCR instrument, selected detection fluorescence is FAM, and gathers fluorescence data when PCR circulation second goes on foot 60 ℃.On LightCycler quantitative PCR instrument, the fluorescence acquisition mode is set at " SINGLE ", and the instrument detecting channel selecting is the F1 passage, and gathers fluorescence data when PCR circulation second goes on foot 60 ℃, and other are default value.

8. interpretation of result

1.LightCycler: F1/F2 reads the result with the fluorescence display format.The baseline setting principle is as the criterion with the vertex just above the blank amplification curve, uses automatic search key value then, when the typical curve relation conefficient greater than 0.99 the time, this reaction is considered as effectively.

2.RG3000: the baseline setting principle is as the criterion with the vertex just above the blank amplification curve, also can adopt automatic search key value, when the typical curve relation conefficient greater than 0.99 the time, this reaction is considered as effectively.

3.ABI Prism 7000: the baseline setting principle is as the criterion with the vertex just above the blank amplification curve, also can adopt automatic search key value, when the typical curve relation conefficient greater than 0.99 the time, this reaction is considered as effectively.

4. after baseline was set, analysis software can provide the copy value of each sample automatically.

5. calculate ABP/SYB ratio.

(2) result

Operation proves that at gene copy number be 10 as stated above ⁴-10 ⁸In the scope, ABP and SYB reagent all can provide linear result, the coefficient R of its typical curve＞0.999 (Fig. 6).

Embodiment 5: the detection specificity analysis of real-time quantitative PCR reagent

(1) materials and methods

A. wild-type and mutant exons 1 9DNA template

Through the wild plasmid of gene sequencing confirmation and 6 types mutant plasmid (Fig. 4).To exons 19 transgenations of other types, carry out synthetic (table 8) according to its base sequence.

EGFR exons 19 base sequences of table 8 synthetic

Numbering	Mutation type	Base sequence (5 '-3 ')
			W	Wild-type	gga ctc tgg atc cca gaa ggt gag aaa gtt aaa att ccc gtc gct atc aag gaa tta aga gaa gca aca tct ccg aaa gcc aac aag gaa atc ctc
M1	Ddel E746- A750(1)	gga ctc tgg atc cca gaa ggt gag aaa gtt aaa att ccc gtc gct atc aag cca tct ccg aaa gcc aac aag gaa atc ctc
			M3	del E746-A750ins T	gga ctc tgg atc cca gaa ggt gag aaa gtt aaa att ccc gtc gct atc aca aca tct ccg aaa gcc aac aag gaa atc ctc
M4	del E746-A750ins P	gga ctc tgg atc cca gaa ggt gag aaa gtt aaa att ccc gtc gct atc aaa aca tct ccg aaa gcc aac aag gaa atc ctc
			M5	del E746-A750ins I	gga ctc tgg atc cca gaa ggt gag aaa gtt aaa att ccc gtc gca att

		aag ata tct ccg aaa gcc aac aag gaa atc ctc
			M6	del E746-T751	gga ctc tgg atc cca gaa ggt gag aaa gtt aaa att ccc gtc gct atc aaa tct ccg aaa gcc aac aag gaa atc ctc
M7	del E746-T751ins I	gga ctc tgg atc cca gaa ggt gag aaa gtt aaa att ccc gtc gct atc aaa att ccg aaa gcc aac aag gaa atc ctc
			M8	del L747-E749	gga ctc tgg atc cca gaa ggt gag aaa gtt aaa att ccc gtc gct atc aag gaa gca aca tct ccg aaa gcc aac aag gaa atc ctc
M11	del L747-T751	gga ctc tgg atc cca gaa ggt gag aaa gtt aaa att ccc gtc gct atc aaa tct ccg aaa gcc aac aag gaa atc ctc
			M12	del L747-T751insP	gga ctc tgg atc cca gaa ggt gag aaa gtt aaa att ccc gtc gct atc aag gaa cct ccg aaa gcc aac aag gaa atc ctc
M13	del L747-T751ins V	gga ctc tgg atc cca gaa ggt gag aaa gtt aaa att ccc gtc gct atc aag gta tct ccg aaa gcc aac aag gaa atc ctc
			M14	del L747-T751ins A	gga ctc tgg atc cca gaa ggt gag aaa gtt aaa att ccc gtc gct atc aag gca tct ccg aaa gcc aac aag gaa atc ctc
M16	del L747-S752ins V(1)	gga ctc tgg atc cca gaa ggt gag aaa gtt aaa att ccc gtc gct atc aag gtt ccg aaa gcc aac aag gaa atc ctc
			M18	del L747-S752ins A	gga ctc tgg atc cca gaa ggt gag aaa gtt aaa att ccc gtc gct atc aag gct ccg aaa gcc aac aag gaa atc ctc
M19	del L747-S752ins D	gga ctc tgg atc cca gaa ggt gag aaa gtt aaa att ccc gtc gct atc aag gat ccg aaa gcc aac aag gaa atc ctc
			M20	del L747-S752ins Q(1)	gga ctc tgg atc cca gaa ggt gag aaa gtt aaa att ccc gtc gct atc aag gaa caa ccg aaagcc aac aag gaa atc ctc
M21	del L747-S752ins Q(2)	gga ctc tgg atc cca gaa ggt gag aaa gtt aaa att ccc gtc gct atc aag gaa cag aaa gcc aac aag gaa atc ctc
			M22	del L747-S752ins S	gga ctc tgg atc cca gaa ggt gag aaa gtt aaa att ccc gtc gct atc aag gaa tcg aaa gcc aac aag gaa atc ctc

B. quantitative PCR detection

In the gradient analysis experiment, adopt synthetic wild-type and del E746-A750 (1) mutant DNA fragment, adjust DNA concentration respectively to 5000pg-0.5pg/ μ l.Respectively get 2 μ l DNA and carry out quantitative PCR detection.

In other experiment, the plasmid DNA fragment concentrations all is adjusted to 5pg/ μ l, and synthetic DNA concentration all is adjusted to 0.5pg/ μ l.Respectively get 2 μ l DNA and carry out quantitative PCR detection (method is the same).

(2) result

Fig. 7 A result shows that the wild-type dna profiling is between 10ng-1pg concentration, and the fluorescent signal and the template concentrations of quantitative PCR reaction are linear.But del E746-A750 (1) the mutant DNA template of same concentrations all fails to detect fluorescent signal in PCR reaction.

Amplified production is carried out taking the photograph sheet behind the electrophoresis, confirm no matter be wild-type or mutant template, band (Fig. 7 B) clearly behind pcr amplification, all occurs.

Taqman probe in detecting wild-type exons 19 content are adopted in the The above results prompting, and Syb Green I DNA dyestuff detects the amplified production total content, can differentiate wild-type and mutant exons 19 by the ratio (ABP/SYB) that calculates the two.In order to verify this point, the present inventor selects 4 kinds of plasmids shown in Figure 4 to detect, and table 9 result shows that the ABP/SYB value of mutant exon all significantly is lower than wild-type.

Further use this method and detect 16 kinds of dissimilar exons 1s 9 transgenations, prove that the ABP/SYB value of all types mutant exon all significantly is lower than wild-type (table 10).

Above presentation of results is used this test kit and can be detected present known all types exons 19 transgenations.

Table 9 quantifying PCR method is to the detection specificity of 4 kinds of EGFR exons 19 transgenations

The plasmid numbering	Mutation type	ABP/SYB ratio		On average
					Experiment one	Experiment two
		201T	Wild-type				0.77	1.27	1.02
693	Wild-type			0.97	0.82	0.90
		694	Wild-type				0.76	0.78	0.77
864	Wild-type			0.81	0.80	0.81
		864-7	del E746-A750(2)				0	0	0
201	del L747-A750ins P			0.31	0.23	0.27
		251	del L747-S752				0.01	0	0.01
694-1	del L747-S752ins V (2)			0	0	0

Annotate: plasmid DNA concentration is adjusted to 5pg/ μ l, gets 2 μ l DNA and carries out quantitative PCR detection.

Table 10 quantifying PCR method is to the detection specificity of 16 kinds of EGFR exons 19 transgenations

Numbering	Mutation type	ABP/SYB ratio		On average
					Experiment one	Experiment two
		W	Wild-type				0.72	0.67	0.70
M3	del E746-A750ins T			0	0	0
		M4	del E746-A750ins P				0	0	0
M5	del E746-A750ins I			0	0	0
		M6	del E746-T751				0	0	0
M7	del E746-T751ins I			0	0	0
		M8	del L747-E749				0	0	0
M11	del L747-T751			0	0	0
		M12	del L747-T751insP				0	0	0
M13	del L747-T751ins V			0	0	0
		M14	del L747-T751ins A				0	0	0
M16	del L747-S752ins V(1)			0	0	0
		M18	del L747-S752ins A				0	0	0
M19	del L747-S752ins D			0	0	0
		M20	del L747-S752ins Q(1)				0	0	0
M21	del L747-S752ins Q(2)			0.01	0.01	0.01
		M22	del L747-S752ins S				0	0	0

Annotate: dna fragmentation concentration is adjusted to 0.5pg/ μ l, gets 2 μ l DNA and carries out quantitative PCR detection.

Embodiment 6: the detection sensitivity analysis of real-time quantitative PCR reagent

(1) materials and methods

A. the concentration with wild-type (693T) and mutant (693-3) plasmid is adjusted to 5pg/ μ l respectively, in different ratios the two is mixed then, makes the content of mutant DNA account for the 0-100% (table 11) of wild-type DNA.Respectively get 2 μ l and carry out real-time quantitative PCR detection (method is the same).

B. selected 2 routine mutants and 2 routine wild-type cancerous lung tissues at random, genomic dna is carried out serial dilution, concentration range is 20ng/ μ l-0.31ng/ μ l, respectively gets 2 μ l and carries out real-time quantitative PCR detection (method is the same).

(2) result

Table 11 result shows, when mutated genes accounts for 10% in the sample DNA, adopt quantifying PCR method it can be detected and since the highest resolution of gene sequencing is a mutated genes account for the DNA total amount 10% or more than, thereby the susceptibility of quantitative PCR detection equates with gene order surveying method.

Table 11 real-time quantitative PCR is to the detection sensitivity of mutated genes

Mutant DNA content	ABP/SYB ratio		Mean value
				Experiment
	1	Experiment 2
100％			0	0	0
	80％	0.095				0.288	0.192
60％			0.281	0.292	0.287
	40％	0.561				0.607	0.584
20％			0.827	0.805	0.816
	10％	0.876				0.911	0.894
5％			0.942	0.899	0.921
	0％	0.890				1.049	0.970

Embodiment 7: the quantitative PCR detection of EGFR exons 19 transgenations in the tissue

(1) material and method

Up to now, the present inventor has finished EGFR exons 19 gene sequencing of 176 routine cancerous lung tissues.In these cancerous lung tissues, select 20 routine mutant tissues at random, the male sex's 16 examples wherein, women's 4 examples, the age is 38-74 year, average out to 59.7 years old, types of organization is gland cancer 7 examples, squama cancer 10 examples, adenosquamous carcinoma 3 examples.In contrast, the present inventor has selected the 20 routine wild-type cancerous lung tissues (table 12) that age, sex and types of organization mate substantially.The genomic dna of each case is diluted to 25ng/ μ l, respectively gets 2 μ 1 and carry out real-time quantitative PCR detection (method is the same).

(2) result

Table 12 result shows that the ABP/SYB value scope of 20 routine wild type tissues is 0.807-2.748, average out to 1.326 ± 0.584.And the ABP/SYB value scope of 20 routine mutant tissues is 0.171-0.789, average out to 0.540 ± 0.201.Difference between two groups has statistics utmost point significant difference (P＜0.001).

Experimental result explanation, with ABP/SYB ratio＜0.8 as threshold value, adopt the real-time quantitative PCR technology can the quick diagnosis cancerous lung tissue in the genetically deficient sudden change of EGFR exons 19.

The quantitative PCR detection of table 12 wild-type and mutant egf R gene extron 19

Mutant					Wild-type
										Numbering	Sex	Age	Type	ABP/SYB	Numbering	Sex	Age	Type	ABP/SYB
59	The man	60	Squama	0.789	65	The man	60	Squama	0.865
										61	The man	60	Glandular scale	0.777	66	The man	60	Glandular scale	1.246
231	The man	55	Gland	0.667	224	The man	55	Gland	1.074
										240	The woman	57	Glandular scale	0.234	250	The woman	56	Glandular scale	1.374
251	The man	50	Gland	0.616	C32	The man	53	Gland	1.045
										C8	The man	65	Gland	0.675	76	The man	64	Gland	1.224
C16	The man	74	Squama	0.402	C48	The man	71	Squama	1.000
										C18	The man	52	Squama	0.288	C35	The man	53	Squama	2.583
C19	The man	67	Squama	0.273	C45	The man	65	Squama	0.807
										C20	The woman	54	Glandular scale	0.758	C5	The woman	57	Glandular scale	1.042
C24	The man	71	Gland	0.363	31	The man	72	Gland	1.182
										C25	The man	55	Squama	0.584	254	The man	55	Squama	1.124
C26	The man	68	Gland	0.554	18	The man	67	Gland	1.196
										C27	The man	56	Squama	0.713	232	The man	56	Squama	0.954
C28	The man	68	Gland	0.171	12	The man	66	Gland	1.059
										C34	The woman	69	Squama	0.400	77	The woman	64	Gland	1.036
C37	The woman	51	Squama	0.634	C50	The woman	54	Squama	2.748
										C38	The man	38	Gland	0.438	C7	The man	28	Gland	1.421
C39	The man	71	Squama	0.690	26	The man	70	Squama	0.952
										C53	The man	53	Squama	0.774	C35	The man	53	Squama	2.583
On average		59.7	0.540±0.201		On average		59.0	1.326±0.586

Although the invention describes concrete example, having a bit is significantly to those skilled in the art, promptly can do various variations and change to the present invention under the premise without departing from the spirit and scope of the present invention.Therefore, claims have covered all these changes within the scope of the present invention.It is for referencial use that this paper is all included in all publications, patent and the patent application that this paper quotes in.

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4.Paez JG, Janne PA, Lee JC waits EGFR mutations in lung cancer:Correlationwith clinical response to gefitinib therapy.Science, and 2004,304:1497-1500

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Sequence table

＜110〉Qinuo Tumour Biology High and New Technology Co., Ltd., Shanghai

＜120〉real time quantitative PCR detecting reagent kit and the method for epidermal growth factor receptor exon 19 deletion mutations

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Claims (7)

1. one kind is detected the test kit of epidermal growth factor receptor exon 19 deletion mutations by real-time quantitative PCR, it is characterized in that described test kit is made up of following:

(a) epidermal growth factor receptor exon 19 gene standard substance;

(b) first reagent set, this reagent set contains a pair of primer and Taqman probe, described primer is to being that ATP to epidermal growth factor receptor exon 19 codings has specific primer in conjunction with the capsule sequence, described Taqman probe has corresponding to 19 the 746th nucleotide sequences to the encoding sequence of 752 bit codons of wild-type epidermal growth factor receptor exon, its 5 ' end links to each other with the fluorescence report group, and 3 ' end links to each other with the photoluminescence quenching group;

(c) second reagent set, this reagent set comprise a pair of primer and DNA dyestuff, and the primer in wherein said primer pair and first reagent set is to identical.

2. test kit according to claim 1,2358 to 3729 nucleotide sequences that wherein said epidermal growth factor receptor exon 19 gene standard substance have people's epithelial growth factor receptor cDNA sequence X 00588.

3. test kit according to claim 1, a pair of primer in wherein said first reagent set have the nucleotide sequence shown in SEQ ID NO:5 and the SEQ ID NO:6 respectively.

4. test kit according to claim 1, wherein said DNA dyestuff are Syb Green I.

5. test kit according to claim 1, wherein said Tagman probe has the nucleotide sequence shown in the SEQ ID NO:7.

6. test kit according to claim 1, wherein said two reagent set all contain warm start Taq enzyme.

7. method by epidermal growth factor receptor exon 19 deletion mutations in the real-time quantitative PCR test sample, this method comprises, (a) with described first reagent set of claim 1 sample is carried out real-time quantitative PCR and detect, obtain the gene copy number of wild-type epidermal growth factor receptor exon 19 in the sample; (b) with described second reagent set of claim 1 sample is detected, obtain total copy number of amplified production in the sample; Whether there are epidermal growth factor receptor exon 19 deletion mutations according to step (a) with in the ratio in judgement sample of the copy number that (b) obtains.

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