CN104328164A - Kit for detecting human EGFR gene mutation by using fluorescence probe hybridization method - Google Patents

Abstract

The invention relates to the field of biology technology and medical science, and concretely relates to a kit for detecting human EGFR gene mutation by using a fluorescence probe hybridization method. Combination of taqman probe, PNA probe and a multiple PCR method is creatively used for detecting EGFR mutation, all operations are finished in a tube, the operation is simple, cover-opening operation is not needed, and generation of pollution is avoided. TM value is used for determining whether mutation happens. A special solid-form storage manner is provided, and transportation and storage are substantially facilitated. The kit is mainly applied to diagnose whether iressa/tarceva and other molecule target medicines are suitable for non-small cell lung cancer patients.

Description

Fluorogenic probe hybridzation method detects Human epidermal growth factor receptor transgenation test kit

Technical field

The present invention relates to biotechnology and medical field, be specifically related to a kind of test kit using hybridization probe to detect EGFR gene mutation site.

Background technology

Nonsmall-cell lung cancer (NSCLC) is worldwide quite general, cause the individual most important reason because of cancer mortality of men and women, chemotherapy seems helpless for treatment NSCLC, patient's curative effect of nearly 85% is not remarkable, in the case, molecular targeted agents receives publicity as new therapy.EGF-R ELISA (EGFR) is a kind of tyrosine protein kinase, process LAN and (or) undergoing mutation in many tumours, control tumor growth by signal transduction, and generate with new vessel, the Infiltration and metastasis etc. of tumour has close relationship.In nonsmall-cell lung cancer, EGFR is overexpression often, but, how many expression of EGFR can not become a level of signification of predict drug response, and research confirms that the sudden change of EGFR is very important for drug reaction, and the sudden change of different exon will cause conformational change, change and strengthen the activity of albumen, same enhancing is to the susceptibility of TKI, and Iressa and Erlotinib are used by U.S. FDA approval as molecular targeted therapy, have significant curative effect to the patient of 10% ~ 15%.KI by with ATP competition binding EGFR Bao Nei Tyrosylprotein kinase district, stop tyrosine residues phosphorylation, thus stop the conduction of EGFR signal path, finally reach a series of tumour cell biological activities such as Tumor suppression propagation, transfer, vasculogenesis.By detecting the catastrophe of patients with lung adenocarcinoma EGFR, and and Clinical symptoms between relation, most suitable patient can be screened and carry out targeted therapy targetedly and provide foundation, to avoid non-rational use of drug.

The EGFR detection method of gene mutation generally applied at present is restriction small segment length polymorphism analysis method (RFLP method) and DNA direct Sequencing, Taqman hydrolysis probes detection etc.RFLP method is the method combined with restriction enzyme digestion by PCR.But the method exists following shortcoming: RFLP experimental implementation is loaded down with trivial details, and sense cycle is long, with high costs, there is first round enzyme and cut the false positive not exclusively caused, non-stopped pipe operation, is easy to pollute, is difficult to meet clinical detection requirement.The principle of DNA direct Sequencing is: it is longer that the method exists following shortcoming sense cycle, both expensive, and non-stopped pipe operation, be difficult to avoid crossed contamination, flux is not high.The sensitivity of DNA direct Sequencing is lower, the problems such as the existence of heterozygous mutant, GC enrichment region make to be difficult to obtain accurate data by once sequencing, need repeatedly to repeat order-checking and just may avoid false positive etc., therefore direct Sequencing method difficulty is applicable to clinical detection.

Taqman hydrolysis probes method uses amplification refractory mutation system (amplification refractory mutation system, ARMS) be combined with fluorescent probe and detect sudden change, the Taq DNA used when increasing according to PCR in theory can not revise primer 3 ' and hold base mispairing, as long as primer 3 ' is held with template 1 base unpaired, just specific product cannot be amplified.For this characteristics design primer, saltant type is increased, wild-type cannot increase, thus is exaggerated mutant signal, is convenient to detect.Utilize ARMS primer pair mutated target sequence to carry out PCR amplification, Taqman probe carries out specific position detection to amplified production, and specific sudden change is identified on Real-time PCR basis.The method existing defects: last base of ARMS technology primer does not mate the amplification can not blocking wild-type DNA completely, in this way there is false-positive risk, and taqman needs to be in charge of operation in conjunction with arms detection EGFR, dna to be measured like this needs to reach certain amount, and troublesome poeration.

Taqman probe generally uses as hydrolysis probes, and use in single mutational site is detected like this and comparatively extensively send out, but relate to multiple mutation detection, then detection site number is subject to passage restriction.Taqmen probe is mentioned as the rarely seen people of having of hybridization probe, it is generally acknowledged Taqman probe in free state and be attached to template time not luminous, only just luminous when nuclease hydrolysis probe.And study confirmation through us, taqman probe is very low at free state fluorescent value, and when being attached to template, linear state is in due to the spirane structure of double-strand, excite group and quenching group due to certain distance so apart, and make the light exciting group to send not by complete cancellation, and this characteristic makes taqman probe can use as hybridization probe.

Multiple asymmetric PCR (multiplex asymmetric pcr, MAP) asymmetric amplification is carried out to multiple target spot, amplified production can be directly used in hybridization and not need through strand sepn process, MAP comprises special primer design method, this design of primers makes different primers have more wide in range annealing temperature, annealing temperature normalization method when being many primer amplifications like this, significantly increases the success ratio that multidigit point increases simultaneously.Current multiple asymmetric PCR is used for fluorescence platform and has no patent report.

Peptide nucleic acid(PNA) (Peptide Nucleic Acid, PNA) be that to grow up the nearly more than ten years take neutral amide bonds as thymus nucleic acid (the Deoxyribonucleic Acid of skeleton, DNA) analogue, its structure is between polypeptide and DNA.PNA can be hybridized with DNA or RNA specifically, forms stable complex body.PNA due to himself feature can to DNA replication dna, genetic transcription, translation etc. have for regulation and control, simultaneously substantially increase genetics as hybridization probe and detect and the efficiency of medical diagnosis and sensitivity.Because PNA can be combined specifically with DNA and RNA, can PNA probe be prepared. compared with DNA probe, stability and the specificity of its hybridization increase greatly.

Summary of the invention

The use taqman probe of the invention, PNA probe are combined with a kind of multiple PCR method and detect EGFR and suddenly change, and all testing processes do not need operation of uncapping, and avoid the generation of pollution.Use TM value to judge sudden change, and all detections complete in a pipe, simple to operate.Provide special solid form storing mode, greatly facilitate transport and store.

Be specially:

1. optimize the usage policy of multiple asymmetric PCR (MAP) at fluorescence platform, MAP applying for a patent (201110448444.8) see us, for fluorescence platform, we optimize below whole system.Add magnesium ion concentration to 6mM, add the tetra-sodium that Pyrophosphate phosphohydrolase comes to produce in suppression PCR process, add the amplification ability that amplification strategy strengthens whole system.

2., for No. 18 and 21 exon mutant nucleotide sequences design Taqman probe, design special MAP primer for conserved sequence.For 18 exons, this test kit can detect G719S/A/C sudden change, and path setting used is FAM passage.For 21 exons, this test kit can detect the sudden change of L858R and L861Q, and passage used is RED610 passage.For these point mutation, we set the term of reference of Tm value.

3. for the feature that 19 exon mutantional hotspots are many and concentrated, we devise special PNA probe and are used as the use of retardance probe, at upstream conservative region design forward primer, at downstream conservative region design taqman probe and negative sense primer, when template is wild-type, its base mates PNA probe completely, because PNA can not by Exonucleolytic enzymic hydrolysis, the carrying out of PNA probe retardance PCR reaction, now will not have amplified production, can't detect signal; And when undergoing mutation, probe Tm value reduces greatly, PNA probe is dissociated from template strand, and pcr reaction is normally carried out, and like this, amplification strand out can use Taqman hybridization probe to detect, and passage used is FAM passage.This test kit can detect any deletion mutantion on 19 exons on E746-T751, judges whether to there occurs sudden change according to or without solubility curve, but can not distinguish concrete sudden change.

4. for 20 exons, we devise conservative primer pair and two probes to detect mutational site, can detect S768I and T790M sudden change, and the insertion mutation of 20 exons, and sense channel used is JOE wavelength channel.For said mutation type, we set the term of reference of Tm value.

5. test kit described in is made up of PCR reaction solution and primed probe and quality control product.PCR reaction solution comprises Kcl, tris-cl, dNTPs, Pyrophosphate phosphohydrolase, warm start polysaccharase, MgCl2, trehalose formation.Primed probe comprises: Map primer pair, Taqman probe, PNA probe etc.Quality control product comprises negative quality control product and positive quality control product is formed.During each component reaction, final concentration is:

Kcl：40~60mM；

Tris-cl: 500~700nM；

dNTPs: 200~600mM ；

MAP primer pair final concentration is often kind of 150 ~ 350nM;

Template DNA 0.01 ~ 10ng/ μ L;

TaqDNA polysaccharase 0.01 ~ 1.0U/ μ L;

MgCl2 final concentration is 1.5 ~ 6.0mM;

Final concentration often planted by Taqman probe is 50 ~ 200nM;

PNA probe concentration is 10 ~ 50nM;

Trehalose final concentration is 10 ~ 60mM;

6. this test kit provides alternative special solids package form, and its content comprises:

UNG enzyme, warm start polysaccharase, Pyrophosphate phosphohydrolase, Tris-Hcl, KCl, dUTPs(AGCU), MgCl2, glycerine, DMSO, trehalose, MAP primer, Taqman probe, Pna probe and tackiness agent.

In order to reach appeal object, its concrete kit method is:

1, design of primers: design of primers uses the conservative region design P3 section primer being published in EGFR nucleotide sequence on NCBI, then P2 section and P1 section primer is designed, wherein replace hypoxanthine base with alphabetical n, hypoxanthine base can be matched with four kinds of base perfections.EGFR primer table is as follows:

2, probe design: according to EGFR particular sequence designing nucleic acid probe: wherein Taqman probe modifies probe through fluorophor, and PNA probe is without modification.

  

3, Tm reference value setting: for 18 exons, be 60.6 DEG C without mutation T m value, if Tm value deviate from more than 0.5 DEG C, then thinks that the one that there occurs in G719S/A/C is suddenlyd change.For the deletion mutantion that 19 exons occur, if there is solubility curve, then thinks and there occurs deletion mutantion; For 20 exons, p20-1 reference value is 71.3 DEG C, if deviate from more than 0.5 DEG C, then thinks that 768 amino acid sites there occurs insertion mutation; P20-2 reference value is 59 DEG C, if depart from more than 0.5 DEG C, then thinks and there occurs T790M sudden change.For 21 exons, probe P21 reference value is 78 DEG C, if depart from more than 0.5 DEG C, then thinks and there occurs L858R or L861Q sudden change.

4, solid reactant preparation: the preparation of reaction system and solid reactant: it is 0.4 μM that every person-portion reactant comprises that forward primer often plants, negative sense primer concentration is 10 μMs, often kind of concentration and probe concentration is 100nM, 2ul dUTPs (2.5 mM of each dUTP), 5mM MgCl2,1.5U warm start polysaccharase, 0.2U Pyrophosphate phosphohydrolase, 0.2U UNG enzyme, 10% trehalose, the glycerine of 1%, above mixed solution low-temperature freeze drying.

5, the preparation of positive reference material and negative reference product: the mutant fragments of 18/19/20/21 exon be inserted in pMD-T vector with primer pair increases.PCR reaction system is 50ul, comprises l0x buffer, 5.0ul; 25mM MgCl2,6.0 ul; L0mM dNTP, 0.5ul; The biotin labeled primer mixture of l00uM, 0. 5ul; DdH2O, 35. 4ul; UNGase (IU/ul), 0.2ul; Taq DNA polymerase, 0.4ul; Template DNA, 2.0ul; 50ul altogether.PCR response procedures is: first 37 DEG C, 5 minutes, 95 DEG C of sex change 15 minutes, then 95 DEG C, 30 seconds; 58 DEG C, 30 seconds; 72 DEG C, 50 seconds; Carry out 40 circulations, last 72 DEG C extend 5 minutes.Above-mentioned PCR primer is through rubber tapping purifying.Prepare positive reference substance 1 (delE746-A750), positive reference substance 2 (delL747-P753insS), negative controls 1 (exon19 wildtype) altogether; Positive reference substance 3 (L858R), positive reference substance 4 (L861Q), negative controls 1 (exon21wild type); 20 ± 3 DEG C of lucifuges, sealed storage, avoid multigelation, validity period be from produce from 1 year.

6, nucleic acid extraction: ordinary method extracts the nucleic acid obtained from patient tissue, its samples sources comprises peripheral blood, patient's pathological tissues, and with paraffin mass, paraffin section, frozen section etc. that these tissues are made, comprises post method and extract and magnetic bead extracting method.

7, Q-PCR reaction system and response procedures:

95℃ 10min

95 DEG C of 30 s, 53 DEG C of 90s 68 DEG C of 1min, 15 circulations

95 DEG C of 30 s, 58 DEG C of 90s 68 DEG C of 1min, 45 circulations

45 DEG C are done solubility curve to 95 DEG C, collection per second 5 fluorescence.

  

Accompanying drawing illustrates:

Fig. 1: show design of primers conceptual scheme, whole primer is divided into syllogic to design, and wherein P2 section base is xanthoglobulin, can be positioned at primer header, afterbody, or middle;

Fig. 2: show MAP amplification schematic diagram, in this test kit, the amplification for each site only only used pair of primers;

Fig. 3: the amplification schematic diagram showing 18 exons, uses FAM passage;

Fig. 4: the probe hybridization figure showing 19 exons, use FAM passage;

Fig. 5: the probe P20-1 hybridization figure showing 20 exons, use Hex passage;

Fig. 6: the probe P20-2 hybridization figure showing 20 exons, use Hex passage;

Fig. 7: the probe hybridization figure showing 21 exons, use red610 passage;

Fig. 8: the detected result figure showing a routine sample.

specific embodiments: clinical flesh tissue pattern detection.

The first step: the extraction of sample DNA, this extracting method is from the method in the biochemical company limited DP322 of sky root.

1. process material:

By the sample cotton swab transposition that wipes across in 2ml centrifuge tube, with scissors, cotton swab part is cut from its bar, add 400 μ l damping fluid GA.Vibrate 15 seconds, room temperature places 5 minutes.

2. add 20 μ l Proteinase K solution, vortex mixing in 10 seconds, places 60 minutes for 56 DEG C, and vortex mixing in every 15 minutes therebetween for several times.

3. add 400 μ l damping fluid GB, fully put upside down mixing, place 10 minutes for 70 DEG C.

4. add 200 μ l dehydrated alcohols, fully put upside down mixing, brief centrifugation.

5. previous step gained solution and flocks are all added (adsorption column CR puts into collection tube) in an adsorption column CR, centrifugal 30 seconds of 12,000 rpm (~ 13,400 × g), outwell the waste liquid in collection tube, adsorption column CR is put back in collection tube.

6. please first check whether add 500 μ l damping fluid GD(uses in adsorption column CR before and added dehydrated alcohol), centrifugal 30 seconds of 12,000 rpm (~ 13,400 × g), outwells the waste liquid in collection tube, is put back in collection tube by adsorption column CR.

7. please first check whether add 700 μ l rinsing liquid PW(uses in adsorption column CR before and added dehydrated alcohol), centrifugal 30 seconds of 12,000 rpm (~ 13,400 × g), outwells the waste liquid in collection tube, adsorption column CR is put back to collection tube.

8. in adsorption column CR, add 500 μ l rinsing liquid PW, centrifugal 30 seconds of 12,000 rpm (~ 13,400 × g), outwells the waste liquid in collection tube.

9. put back in collection tube by adsorption column CR, centrifugal 2 minutes of 12,000 rpm (~ 13,400 × g), outwells waste liquid.Adsorption column CR room temperature is placed several minutes, thoroughly to dry rinsing liquid remaining in sorbing material.

10. proceeded to by adsorption column CR in a clean centrifuge tube, to the unsettled dropping in adsorption film mid-way 20-50 μ l elution buffer TB, room temperature places 2-5 minute, 12,000 rpm (~ 13,400 × g) centrifugal 2 minutes.

The sample DNA of 11. preparations is stand-by.

Second step: pcr amplification

The PCR solid reactant of freeze-drying in test kit is added ultrapure water 10 μ l by 1, adds DNA profiling, adds water and complement to 20ul.

2 response procedures are:

95 ℃ 15min

95 DEG C of 30 s, 53 DEG C of 30s 72 DEG C of 50S, 15 circulations

95 DEG C of 30 s, 76 DEG C of 90s 45 circulations

45-95 DEG C of solubility curve collection fluorescence per second 5 times.Channel selecting 480,568,610,710 passages.

3rd step: contrast each channel reference Tm value scope, determine whether to undergo mutation.Sudden change only can be thought in scope.If Tm value is near term of reference border, then loading errors should be considered.Do result as repeated still consistent, then follow reference.

  

Result: detect 197 routine nonsmall-cell lung cancer clinical samples, finds 71 sudden changes, and wherein 2 patients have two sudden change.

Concrete outcome is as follows:

Exon	Mutation type	Sequencing	This examination box detects	Number
					18	Missense mutation	G719S	G719S	1
		G719A	G719A	1
							G719S	G719S	1
19	Disappearance/insertion mutation	K745_D749del	745-751del	1
							E746_A750del	745-751del	19
		E746_A750delinsF	745-751del	1
							E746_A750del	745-751del	9
		E746_A750del	745-751del	1
							E746_A750delinsV	745-751del	2
		E746VinsPVAIKE	745-751del	1
							L747_D749delinsP	745-751del	1
		L747_T751delinsP	745-751del	1
							L747_P753delinsQ	745-751del	1
		L747_T751del	745-751del	2
							L747_P753delinsS	745-751del	6
				1
					20	Wrong meaning/insertion mutation	S768I	S768I	3
		A767_V769del	Insertion mutation	1
							D770_H773insGSVD	Insertion mutation	1
		N771_P772insGY	Insertion mutation	1
							H773_R776insYNPY	Insertion mutation	1
		C775_R776insPA	Insertion mutation	1
					21	Missense mutation	R836C	Do not detect	1
		L858R	L858R	13
						Total			71

This experimental result conforms to sequencing detected result, and the sudden change that test kit can detect all proves by sequencing, illustrates this test kit and has very high susceptibility and specificity.

Conclusion: test kit of the present invention fast, accurately can detect the sudden change of EGFR, is significant for Iressa medication guide.

SEQUENCE LISTING

<110> Shanghai Xingyao Medical Technology Development Co., Ltd.

Shanghai Fosun Pharmaceutical (Group) Co., Ltd.

Liu, great waves

Summer, virtuous

Wu, controls greatly

<120> glimmering pin probe hybridization method detects Human epidermal growth factor receptor transgenation test kit

<130> fluorogenic probe hybridzation method detects Human epidermal growth factor receptor transgenation test kit

<160> 14

<170> PatentIn version 3.3

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Claims (5)

1. the detection method of a human EGFR gene mutations and test kit, it is characterized in that multiple asymmetric PCR increases, PNA/LNA probe as retardance probe, fluorogenic hybridization probe solubility curve judged result and comprise solid form exist 1xPCR reactant (by primer mixture, tris-cl, Kcl, MgCl ₂, warm start polysaccharase, UNG enzyme and lyophilized vaccine composition), its special character is:

A: special syllogic primer, its syllogic design of primers and follow-up amplification are the multiple strand pcr amplification of MAP(of original creation) technology, its sequence is SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8;

B: special probe, 4 selected probes have good specificity and conservative property, can judge sudden change, be respectively SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO. 20 according to the change of Tm value;

C: special storage condition: this test kit is the storage mode that PCR reactant provides solid form;

D: reaction conditions is by optimizing, be roughly divided into two stages, wherein the first stage is concentration stage, 3 primers that this stage working concentration is extremely low and an excessive forward primer, temperature of reaction is generally about 50 DEG C, and amplification stage lower concentration primer will be consumed, and subordinate phase only has the work of negative sense primer subsequently, along with the carrying out of amplification, a large amount of strand object nucleotide sequences will be produced;

E: special retardance probe, this test kit employs special PNA and blocks the expression that probe shields 19 exon wild type genes, substantially increases the accuracy of diagnosis.

2. test kit described in claim 1, primer amplification strategy is two take turns amplification, is divided into double-strand non-symmetric amplification stage and strand asymmetric amplification stage.

3. the test kit in claim 1, its lyophilized vaccine composition is by trehalose, and glycerine and tackiness agent form.

4. the test kit in claim 1, the detection method of its PCR primer is fluorogenic probe hybridzation, dot hybridization, reverse dot blot hybridization, solution hybridization platform.

5. the test kit described in right 1, its purposes is for detecting the sudden change of human EGFR gene.