CN106399518A - Probe for human EGFR genetic mutation detection, kit and detection method thereof - Google Patents

Probe for human EGFR genetic mutation detection, kit and detection method thereof Download PDF

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CN106399518A
CN106399518A CN201610873223.8A CN201610873223A CN106399518A CN 106399518 A CN106399518 A CN 106399518A CN 201610873223 A CN201610873223 A CN 201610873223A CN 106399518 A CN106399518 A CN 106399518A
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CN106399518B (en
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廖玮
莫亚勤
吕佳韩
张晨光
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Guangzhou Easy Living Biotechnology Co Ltd
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Abstract

The invention relates to the field of gene detection, in particular to a probe for human EGFR genetic mutation detection, a kit and a detection method thereof. The probe for human EGFR genetic mutation detection provided by the invention comprises any one or several kinds of probes in 1 to 35 probe pairs. Each probe pair comprises a capture probe and a detection probe; the nucleotide sequences of the 1 to 35 probe pairs are shown as SEQ ID NO: 1 to 70. The probes all use hotspot mutation design on the human EGFR, and are obtained through multi-time screening; the capture probe and the detection probe are used in a matched way; the advantages of high sensitivity, high specificity and accurate and reliable results are realized. The invention also provides a kit for human EGFR genetic mutation detection. The kit only needs few detection samples; in the detection process, the electric field effect is utilized, the reaction speed is accelerated; the reaction time is shortened; the whole detection process can be completed in half an hour; the time required for detection can be greatly saved; in addition, the detection sensitivity and the accuracy are high.

Description

For the probe of Human epidermal growth factor receptor detection in Gene Mutation, test kit and its detection method
Technical field
The present invention relates to field of gene detection, in particular to for the probe of Human epidermal growth factor receptor detection in Gene Mutation, reagent Box and its detection method.
Background technology
The environmental factorss such as air pollution lead to China in Recent Years pulmonary carcinoma number of patients to sharply increase.100000 human lung cancer sickness rate Increase to 63% in 2011 from 39.6% in 2002, the sickness rate of especially Children and teenager is substantially improved.Reform is opened Since putting 30 years, the death toll that pulmonary carcinoma leads to increased 465%, and pulmonary carcinoma has become as sickness rate in China and mortality rate is equal The malignant tumor ranking the first.By traditional histopathologic classification, pulmonary carcinoma can divide small cell lung cancer and nonsmall-cell lung cancer.Non- Small cell lung cancer (Non-small-cell Lung Cancer, NSCLC), including scale cancer, adenocarcinoma, large cell carcinoma, with minicell Pulmonary carcinoma compares the division of its growth of cancer cells relatively slowly, and diffusion transfer is relatively later, and nonsmall-cell lung cancer accounts for the 80- of pulmonary carcinoma sum 85%.
EGF-R ELISA (Epidermal Growth Factor Receptor, EGFR) is epidermal growth factor (EGF) receptor of cell proliferation and signal transduction.EGFR belongs to one kind of ErbB receptor family, and this family includes EGFR (ErbB- 1), HER2/c-neu (ErbB-2), Her 3 (ErbB-3) and Her 4 (ErbB-4).EGFR belongs to tyrosine kinase receptor, EGFR with its part after extracellular region is combined, EGFR intermolecular formation homodimer, also can be combined shape with other HER family molecules Become heterodimer, the tyrosine kinase (TK) of its intracellular region is activated afterwards, the mutual phosphorylation of tyrosine residue, subsequently downstream Coupling protein (PLC γ, aCBL, GRB2, SHC, p85) is specifically bound with this phosphorylation position, passes the signal along to downstream RAS-MAPK path, PI3K-AKT path, STAT path etc., thus the escape of activationa and proliferation, apoptosis, angiogenesiss, transfer Deng with the biological behaviour closely related with tumor development.In all kinds of tumors including pulmonary carcinoma, frequently find The overexpression of EGFR gene, but also relevant with prognosis, therefore EGFR attracts tremendous attention as molecular target.
Research shows, EGFR mutant gene and tyrosine-kinase enzyme level class medicine (tyrosine kinase Inhibitors, TKI) use effective in cure association, these medicines include Iressa (IRESSA) and Tarceva (Tarceva), EGFR genetic mutation affects the clinical efficacy of TKI.EGFR mutation is main to include 4 types：Exons 19 deletion mutation, outer aobvious Sub- 2l point mutation, exons 18 point mutation and extron 20 insertion mutation.Have now been found that the prominent of EGFR genetic mutation more than 90% Become and be located at exons 19,21, modal EGFR sports exons 1 9LREA disappearance and exon 2 1L858R mutation, the two Tyrosine kinase domain all can be led to activate, and be the sensitive mutation of TKI, the T790M of extron 20 is mutated and EGFR- TKI acquired drug-resistance is relevant, and also perhaps eurypalynous mutation clinical meaning is still not clear.Patients with lung adenocarcinoma EGFR gene is sensitive prominent Become positive rate and be about 10% in Caucasia crowd, be 50% about in asian ancestry crowd and China.
EGFR genetic mutation detection at present is broadly divided into detection and the cycling tumor DNA of tumor tissues sample The detection of (Circulating tumor DNA, ctDNA).At present, clinically mainly obtained by modes such as biopsy or operations Tumor tissues sample detecting EGFR genetic mutation, but invasive means need to be adopted due to obtaining tumor tissues specimen, This process often increases the misery of patient, produces extra operation risk, and tumor has heterogeneity, for having sent out For the cancer patient of raw transfer, only take the cancerous tissue at certain position by puncturing or performing the operation, can not reflect that patient is overall Situation.Secondly, some patients situation of itself determines it and is not suitable for biopsy, and some tumors are being punctured or handss After the disturbance of art, there is the risk accelerating transfer.Finally, there are the problems such as expense high latency length in biopsy, its Hysteresis quality is also unfavorable to the treatment of patient.
Therefore, in recent years the concept of " liquid biopsy " is risen, and its basic thought is to replace with body fluid samples such as blood For the detection of tumor tissues sample row pathology, molecular biology, by detecting in patient body fluid sample (mainly blood) Cycling tumor DNA has become as a kind of trend obtaining oncogene abrupt information.Compared with the biopsy of current standard, leather The liquid biopsy of life property has following irreplaceable advantage：Wound is little, repeatable, homogenizing is heterogeneous, real-time judge curative effect, And dynamically adjust Treatment decsion with the development of tumor.Therefore, the year ten of MIT Technology Review issue in 2015 Quantum jump technology (Breakthrough Technologies2015), ASCO year progress (Clinical cancer Advance 2015) in hope to following 10 years, liquid biopsy all appears on the list of successful candidates.Follow the trail of whole disease by detecting ctDNA In journey, the specific gene of tumor changes, and has important value to tumor screening, diagnosis, curative effect monitoring and Index for diagnosis etc., with When can therefrom explore the molecular mechanism of tumour metastasis and recurrence and drug resistance, identify new targeted therapy site etc., the therefore inspection of ctDNA Survey one of the three big hot topic directions having become as tumor liquid biopsy application (note：Another two is CTC and excretion body).
At present, main detection method of gene mutation mainly includes direct sequencing (PCR-sanger sequencing), amplification Check abruptly-changing system (Amplification refractory mutation system, ARMS), denaturing high-performance chromatography Method (Denaturing high-performance liquid chromatograph, DHPLC), high-resolution solubility curve (high resolution melting, HRM), allele specific oligonucleotide Taqman polymerase chain reaction (Competitive Allele-specific Taqman polymerase chain reaction, CAST-PCR), digital pcr (digital PCR, dPCR), high throughput sequencing technologies (High-throughput sequencing, HTS) etc..
1st, direct sequencing.Direct sequencing designs PCR primer first against mutational site, is expanded by PCR and obtains mesh Genetic fragment, then PCR primer is carried out with sanger sequencing and preliminary analyses is carried out to sequencing result.Preliminary analyses complete Afterwards, the PCR primer to the sample that may carry abrupt information, needs to reclaim by cutting the methods such as glue, the PCR primer after recovery is even Connect cloning vehicle (generally plasmid), select positive colony sequencing to confirm to be mutated.
2nd, abruptly-changing system is checked in amplification.The method must with template DNA complementation just using the last bit bases in 3 ' ends of PCR primer Can effective amplification principle, design for the specific PCR amplimer in mutational site, under strict amplification condition, only When primer 3 ' base is matched with template, PCR reacts and just can be normally carried out, thus detecting mutation.Drawn by designing two upstreams Thing, the 3 ' ends of wherein are complementary with normal gene sequence, and the 3 ' ends of another are complementary with mutant gene sequence, during detection respectively Add two kinds of forward primer and shared downstream primer, carry out two parallel PCR, according to two PCR reactions as a result, it is possible to Distinguish with the presence or absence of mutation and sport homozygote or heterozygote.The detection sensitivity of ARMS method is about 1% about.
3rd, denaturing high-performance liquid chromatography.The principle of the method based on occur mispairing heteroduplex DNA with mate completely Homozygosis double-stranded DNA unwind the difference of feature, carry out separating using chromatographic process.Because heteroduplex occurs at mutational site Mispairing, it is easy to form Y-shaped structure, is reduced with the fixing phase binding ability of chromatographic column, therefore heteroduplex DNA is than homozygosis double-strand DNA is preferentially eluted out, and can decide whether there is mutation by the change of eluting peak.The advantage of the method be to known and Unknown mutation all can detect, but still needs to before detection obtain genes of interest fragment by PCR, and detection process needs to open reaction Pipe, easily causes pollution, and detection sensitivity is 5% about, and not can determine that its concrete mutation type for positive findingses, Need for sequence verification eventually.
4th, high-resolution solubility curve.The method is the physical property based on nucleic acid fragment, is incorporated into by saturable dye Pcr amplification product, the solubility curve mutation analysises gene mutation of monitoring PCR primer.The detection sensitivity of the method is about 5% Left and right, not can determine that concrete mutation type for positive findingses, finally needs for sequence verification.
5th, allele specific oligonucleotide Taqman polymerase chain reaction.The method adopts and optimizes Taqman probe, by spy Different in nature MGB probe stops the combination of PCR primer and wild type DNA, therefore can be with preferential amplification mutant DNA, this detection method Sensitivity 1% about.
6th, digital pcr.The nucleic acid quantification that digital pcr is counted based on single-molecule PCR method, is a kind of absolute quantitation Method.Mainly adopt micro-fluidic or microdroplet method, the nucleic acid solution after Macrodilution is dispersed to the microreactor of chip Or in microdroplet, the nucleic acid-templated number of each reactor is less than or is equal to 1.So after PCR cycle, there is a nucleic acid The reactor of molecular template will provide fluorescence signal, does not have the reactor of template just not have fluorescence signal.According to relative scale With the volume of reactor it is possible to extrapolate the nucleic acid concentration of original solution.The detection sensitivity of digital pcr is about 0.1% Left and right.
7th, high throughput sequencing technologies.This technology is that (referred to as generation sequencing technologies) are revolutionary changes to traditional Sanger sequencing Become, its ultimate principle is to be sequenced in synthesis, on the basis of the sequence measurements such as Sanger, with the fluorescent labeling four of different colours Plant different dNTP, when archaeal dna polymerase synthesis complementary strand, often add a kind of dNTP and will discharge different fluorescence, according to The fluorescence signal (such as Illumina microarray dataset) catching, or caught in DNA building-up process by quasiconductor sequence testing chip H+ change (such as Life ion torrent microarray dataset), and through specific software processing, thus obtain treating Survey the sequence information of DNA, once to millions of nucleic acid molecules, sequencing is carried out to hundreds of thousands, be otherwise known as sequencing of future generation Technology (next generation sequencing, NGS).The sharpest edges of this technology are its high flux, can be simultaneously right Multiple samples are sequenced parallel, can also find unknown mutation in addition to known mutations, and detection sensitivity is about in 1-5%. The testing process of high-flux sequence generally comprises DNA extraction and purification, structure library, sequencing template preparation, sequencing, bio information Analysis link.
But, there is following defect in prior art：
Direct sequencing.Direct sequencing has the advantages that to can determine mutational range and type, but because sanger surveys Sequence flux is low, and the restriction of sequence measurement itself leads to its sensitivity not high, and content can only be more than with 20% gene mutation Detected, the needs of practical application far can not be met, especially for heterogeneous tumor somatic mutation, muting sensitivity Substantial amounts of missing inspection will be led to.Meanwhile, sequencing detection complex operation, poor in timeliness, for requiring high-timeliness and high sensitivity Practical application detects, the limitation of sequencing highlights already.
Abruptly-changing system is checked in amplification to be needed using real-time fluorescence quantitative PCR detection technique, and each pair primer can only detect one kind It is mutated, and every kind of mutation needs individually to set up a PCR reaction system, detect operation during multiple samples multiple detection site simultaneously Loaded down with trivial details, and the method needs extracting and purification DNA, larger to the requirement of sample, be not suitable for detecting multiple gene mutation simultaneously Site, and PCR operating process is also easy to produce Aerosol Pollution, brings false positive risk.Allele specific oligonucleotide Taqman polymerase Chain reaction also haves the shortcomings that same.
Denaturing high-performance liquid chromatography and high-resolution solubility curve method and positive findingses not can determine that it is concrete Mutation type, finally needs for sequence verification.The detection sensitivity of two methods is 5% about it is impossible to meet practical application Needs.
Although digital pcr technology sensitivity is higher, this technology equally easily produces false positive results, simultaneously because its behaviour Make complicated, the requirement to experimental implementation is high, this external equipment and reagent is expensive seriously limits its large-scale promotion application. High throughput sequencing technologies need through DNA extraction with purification, structure library, sequencing template preparation, be sequenced several links, and operation is numerous Trivial, the requirement to experimental implementation is high.Equally, expensive equipment and reagent price seriously limit its large-scale promotion application.
In view of this, the special proposition present invention.
Content of the invention
The first object of the present invention is to provide the probe for Human epidermal growth factor receptor detection in Gene Mutation, and these probes are by capturing spy Pin and detection probe composition, capture probe specificity captures the mutational site of Human epidermal growth factor receptor gene, and detection probe detects mutational site Flanking sequence, and then realize the purpose of precisely detection.
The second object of the present invention is to provide Human epidermal growth factor receptor gene mutation detection kit, and this test kit only need to detect on a small quantity Sample, detection process utilizes electric field action, accelerates reaction rate, shortens the response time, whole detection process can be in half an hour Inside complete, greatly save detection required time, and detection sensitivity and accuracy rate height.
In order to realize the above-mentioned purpose of the present invention, spy employs the following technical solutions：
Provided by the present invention for the probe of Human epidermal growth factor receptor detection in Gene Mutation, described probe includes appointing of 1-35 probe centering One or more, each probe centering includes capture probe and detection probe；
SEQ ID NO in the nucleotide sequence of 1-35 probe pair such as sequence table：Shown in 1-70.
The probe that the present invention provides is according to as SEQ ID NO：Wild-type sequence shown in 71-91 and such as SEQ ID NO： The design that mutant sequences shown in 92-126 are carried out successively, wherein, 1-35 probe is to being corresponding in turn in SEQ ID NO：92- Mutant sequences shown in 126, and mutant sequences are SEQ ID NO：92nd, 93,94 corresponding wild-type sequences are as SEQ ID NO：Shown in 71；Mutant sequences are SEQ ID NO：96、97、99、100、101、103、116、117、118、121、122、 123rd, 125 corresponding wild-type sequences are as SEQ ID NO：Shown in 73.Remaining mutant sequences press sequence number successively with open country Raw type sequence pair should.Applicant through lot of experiments, takes into account sensitivity and the specificity of capture probe and detection probe, finishing screen Choosing obtains sensitivity and all good 1-35 probe pair of specificity.1-35 probe to being corresponding in turn in above-mentioned mutant sequences and Wild-type sequence.
Provided by the present invention for the 1-3 probe in the probe of Human epidermal growth factor receptor detection in Gene Mutation to being outer aobvious for EGFR Son 18 point mutation design, to being exons 19 deletion mutation design for EGFR, 29-33 probe is to being to be directed to for 4-28 probe EGFR extron 20 mutation design, 34-35 probe to be for EGFR exon 21 point mutation design.Specifically, 1-3 visits For nucleotide sequence such as SEQ ID NO：Shown in 1-6；The nucleotide sequence of the capture probe of 1 probe centering such as SEQ ID NO：Shown in 1, detection probe such as SEQ ID NO：Shown in 2；The nucleotide sequence of the capture probe of 2 probe centerings such as SEQ ID NO：Shown in 3, detection probe such as SEQ ID NO：Shown in 4.Correspondingly, remaining probe is to by that analogy.
The present invention, through repeatedly screening the capture probe obtaining and detection probe mating reaction, has sensitivity height, specificity By force, the Different categories of samples containing target sequence can be detected, result is accurately and reliably.
In particular it relates to sequence as shown in table 1.
Table 1 sequence
The present invention adopts the change of the signal of telecommunication to realize detection to EGFR genetic mutation site, based on this, further, institute State and be provided with label in detection probe, described label be preferably digoxin, biotin, in marked by fluorescein isothiocyanate thing Any one.So, detection probe is combined with the follow-up enzyme with correspondence markings adding, and provides base for detection change in electric Plinth.
Present invention also offers Human epidermal growth factor receptor gene mutation detection kit, at least contain above-mentioned probe, enzyme and with institute State any one or more in the corresponding substrate of enzyme；
The material being combined with the label of setting in described detection probe is marked with described enzyme.
Preferably, when the label in detection probe is digoxin or Fluorescein isothiocyanate (abbreviation FITC), on enzyme The material of labelling is DigiTAb or Fluorescein isothiocyanate antibody accordingly；When the label in detection probe is biotin When, on enzyme, the material of labelling is Avidin.
I.e. detection probe and enzyme can be by FITC and anti-FITC antibody or digoxin and DigiTAb or Avidin such as chain The mode that mould Avidin is combined with biotin etc. is combined.
Preferably, described enzyme is horseradish peroxidase polymer (abbreviation Poly-HRP) or alkali phosphatase；
When described enzyme is horseradish peroxidase polymer, substrate is TMB and H2O2Mixture, o-phenylenediamine (O- Phenylenediamine, OPD), ABTS [2,2'-azino-di- (3-ethylbenziazobine sulfonate-6)], Any one in 3- (4- hydroxyl)；
Wherein, the product after OPD oxidation is deep orange color or brown, and ABTS product is in aeruginouss.
When described enzyme is alkali phosphatase, substrate is para-nitro-pheneye phosphate (p-nitropheny phosphate Disodium salt, pNPP), the compositionss of BCIP and NBT, 4-NPP, naphthols AS-BI phosphate, naphthols- Any one in AS-MX- phosphate.
Wherein, pNPP generates yellow water-soluble product under the action of alkaline phosphatase；BCIP and NBT is alkaline phosphorus One of optimal chromophoric substrate combination of sour enzyme, product is navy blue；4-NPP, product is yellow soluble；Naphthalene Phenol AS-BI phosphate, product is red insoluble；Naphthols-AS-MX- phosphate, product is red insoluble.
The EGFR genetic mutation detection kit that the present invention provides is convenient for the detection offer of EGFR genetic mutation.
Detect to be more convenient for it is preferable that described test kit also include for by fixture fixing for described capture probe, Hybridization buffer, closed protein, PBS, cleanout fluid, detection orifice plate.
Wherein detection orifice plate concrete structure as disclosed in Application No. 201620769829.2 entitled detecting electrode with And shown in the detection orifice plate in detection orifice plate.
Further, described closed protein be casein, hyclone, any one in calf serum.
Each material in the test kit that the present invention provides can be by individualism it is also possible to presented in mixed liquor.
Further, described fixture includes conducting polymer materials and salts compound；
Described conducting polymer materials are any one in aniline, thiophene and pyrroles's electroconductive molecule monomer；
Described salts compound is chlorate, nitrate, any one in sulfate.
Wherein, salt can be sodium, potassium, magnesium, ammonium etc.；If chlorate can be sodium chloride, potassium chloride, magnesium chloride, chlorination Ammonium etc., similarly, nitrate can be sodium nitrate, potassium nitrate, magnesium nitrate, ammonium nitrate etc., and sulfate can be sodium sulfate, sulphuric acid Potassium, magnesium sulfate, ammonium sulfate etc..
When the EGFR genetic mutation detection kit that the present invention provides is used for detecting, the first step is passed through to occur under current field condition Polyreaction and by capture probe be fixed on detection orifice plate on, for subsequent detection provide basis.Therefore, for the ease of detection behaviour Make it is preferable that described capture probe and described fixture are presented in capturing solution, described capture solution contains following one-tenth Point：The percentage by volume of conducting polymer materials is 0.1%～5%, the concentration of salts compound be 0.01mol/L～ 2mol/L, the concentration of capture probe is 0.5-1.5 μm of ol/L.
I.e. when the detection, directly capture solution is added in the reacting hole of detection orifice plate, in general, 96 In the detection orifice plate in hole, the volume of each reacting hole is 300 μ l, adds the 20-80 μ l capture solution all may be used.
After capture solution adds to the reacting hole of detection orifice plate, carry out electric field treatment so that capturing the composition in solution It is fixed in the reacting hole of detection orifice plate, be beneficial to the carrying out of subsequent reactions.
In order to improve the convenience of detection, further, described capture probe and described fixture are attached to described detection Orifice plate exists；
Specifically adopt following methods preparation：Described capture solution is added in the reacting hole of described detection orifice plate, then Clean after electric field treatment.
During cleaning, program is：From bottom-purge program：The side imbibition of marginal not liquid；Cleaning procedure from top to bottom：First fluid injection, then Imbibition；It is repeated once above-mentioned steps.
The mixed liquor of sample to be tested and hybridization buffer is added, wherein, buffer is in 85-95 DEG C of water-bath for hybridization after cleaning Process 5-15min, then place and be cooled to room temperature, with sample to be detected with volume ratio for 1:1.5-2.5 mixing, then take 20- 80 μ l add in the reacting hole of detection orifice plate, carry out electric field treatment, are then carried out using cleaning step same as described above.
Hybridization buffer in the present invention provides good environment, any hybridization realizing this function for the hybridization of probe Buffer is all within the scope of the present invention.If hybridization buffer used can be any one in following；
Hybridization buffer 1：Containing Ficoll 400 1g/L, PVP360 1g/L, BSA 1g/L, NaH2PO480mM, Na2HPO4420mM, EDTA 2.5mM, SDS 6g/L, NaCl 43.8g/L, Sodium Citrate, usp, Dihydrate Powder 36.9g/L, pH are 7-8；Should Buffer is first heated to 65 DEG C before use.
Hybridization buffer 2：In this solution, the percentage by volume of 20 × SSC is 10%, the volume hundred of 100 × Denhardt Fraction is the mass concentration of 5%, Denatured salmon DNA is 0.1mg/ml, and the percentage by volume of Methanamide is 40%；
Hybridization buffer 3：50% Methanamide, 2 × SSC, 10% dextran sulfate (pH is 7)；
Hybridization buffer 4：The sodium phosphate buffer of 1.5M NaCl, 40mM pH 7.2,10mM EDTA pH 8,10 × Denhardt's solution, 0.2%SDS；
Hybridization buffer 5：20-100nM Tris-HCl, 20-200nM MgCl2, 0.01%-5% dextran sulfate；
Hybridization buffer 6：5 × SSC, 1% closed protein, 0.1%N- Hamposyl L, 0.02%SDS；
Hybridization buffer 7：0.5M sodium dihydrogen phosphate, pH is 7.5；10mM EDTA, 7%SDS.
Percent in above-mentioned hybridization buffer refers both to weight/mass percentage composition, and the content of each component is also each component whole Final concentration in individual solution.
In addition, in the above-mentioned hybridization buffer of the present invention each component content, can have and change by a small margin, this change Within the scope of the present invention.
Further, described detection probe is presented in detecting solution；
Described detection solution is that the wherein percetage by weight of closed protein is 0.1%～5%, detection using PBS as solvent The concentration of probe is 0.5-1.5 μm of ol/L.
In detection solution, closed protein such as caseic effect is non-specific sites in closing detectable substance, to improve detection Susceptiveness and accuracy.
Further, detection solution 20-80 μ l add to detection orifice plate reacting hole in, electric field treatment, then adopt with Above-mentioned identical cleaning step is carried out.
Further, described enzyme is presented in enzyme liquid；
Described enzyme liquid is that the wherein percetage by weight of closed protein is 0.1%～5%, the volume of enzyme using PBS as solvent Percent is 0.05%～0.2%.
In order to close non-specific sites in detectable substance, improve susceptiveness and the accuracy of detection, in enzyme liquid, also contain envelope Close albumen.Correspondingly, the addition of enzyme liquid is 20-80 μ l, incubated at room 20-40min, and detection orifice plate is carried out, and cleans journey Sequence once repeats more than above-mentioned cleaning procedure.
Further, described cleanout fluid include containing 0.1 × SSC-2 for 0.04%-0.06%SDS for the mass concentration × SSC mixed liquor and containing percentage by volume the PBS solution for 0.05%-0.15%Tween20.
PBST (0.1%Tween20) 1L:Add the 20 × PBS of 50ml in the distilled water of 949ml, mix, add 1ml's Tween 20, mixes.
Two kinds of cleanout fluid are adopted, the step before adding enzyme liquid is all to be 0.04%- using mass concentration in the present invention 0.1 × the SSC-2 of 0.06%SDS × SSC mixed liquor is carried out, and is washed with PBST after enzyme due to adding.
By cleaning, nonspecific combination and other unreacted compositions are removed, improves the accuracy of detection.
In addition, the above-mentioned mixed liquor of the present invention is all the concentration of working solution, but according to demand can also be by above-mentioned work The several times of the concentration of liquid are first made into mother solution, dilute accordingly when using again.Therefore, the mother solution of respective concentration is also at this In bright protection domain.
Wherein, 20 × PBS 1L formula is：Potassium dihydrogen phosphate：5.4g, disodium hydrogen phosphate：28.4g, sodium chloride：160g, chlorine Change potassium 4g, plus deionized water about 800mL is sufficiently stirred for dissolving, and is subsequently adding concentrated hydrochloric acid and adjusts pH to 7.4, last constant volume to 1L.Phase Ying Di, PBS are 1 × PBS, and 20 × PBS is diluted 20 times.
Cleanout fluid is also usually SSC and SDS first configuring high concentration, then is diluted accordingly.
The Human epidermal growth factor receptor gene mutation detection kit that the present invention provides, compared with prior art, has concurrently accurate reliable, quick Convenient, economic clear superiority, beneficial to repeated multiple times sampling detection, range is wide, and sampling, to human zero damage, is preferable Liquid biopsy product.
Present invention also offers a kind of method of detection Human epidermal growth factor receptor gene mutation, comprise the following steps：
Above-mentioned capture solution is added in detection orifice plate, electric field treatment 3-10 circulates, the program of electric field treatment is： Voltage 200-500mV, 1-5s；Voltage 800-1500mV, 1-5s；
Electric field treatment is cleaned using 2 × SSC mixed liquor that described mass concentration is 0.04%-0.06%SDS, so after finishing Add the mixed solution of sample to be tested and hybridization buffer afterwards, electric field treatment 3-10 circulates, and the program of electric field treatment is：Voltage 200-500mV, 1-5s；Voltage 300-800mV, 1-5s；
Detection orifice plate cleaning, is subsequently adding above-mentioned detection solution, electric field treatment 3-8 circulates, the program of electric field treatment For：Voltage 200-500mV, 1-5s；Voltage 300-800mV, 1-5s；
Detection orifice plate cleaning, is subsequently adding above-mentioned enzyme liquid, cleans after incubation；
Add substrate, reading under the electric field treatment that voltage is -100～-300mV, obtain current value.
Preferably, described sample to be tested is body fluid, and described body fluid includes but is not limited to blood plasma, saliva, hydrothorax.
The method of the detection Human epidermal growth factor receptor gene mutation that the present invention provides, capture solution adds detection orifice plate, in electric field action Lower generation polyreaction, capture probe is fixed on plate bottom；It is subsequently adding detection sample, capture probe capture target gene mutation Sample；Add detection solution, the detection probe detection mutational site flanking sequence of biotin labeling；Add enzyme liquid, enzyme passes through it The Avidin of labelling is identified with the biotin of detection probe and combines；Add the substrate of enzyme, redox reaction occurs, produce electricity Stream, sensed current signal completes whole detection process.
Compared with prior art, beneficial effects of the present invention are：
(1) easy and simple to handle quick
Prior art is all based on round pcr it is necessary first to extract and purification DNA from specimen, then needs to DNA sample Originally enter performing PCR amplification, NGS is more intended to through DNA extraction and the links such as purification, structure library, sequencing template preparation, sequencing, behaviour Make that step is numerous and diverse, the requirement to operation is high.Therefore, if clinically needing 1-3 days using ARMS-PCR, digital pcr technology Report just can be gone out, even more need 1-2 week just can go out report using NGS technology, take appropriate treatment to arrange for needing to race against time For the tumor disease applied, such report cycle seriously restricts it in clinical application.
The test kit that the present invention provides induces release and measurement (EFIRM) to detect ectosome biomarker by electric field, uses When liquid biopsy, only need a small amount of patient body fluid sample, such as extract peripheric venous blood or the collection a small amount of saliva of patient of patient 1ml Liquid, sample does not need DNA extraction and amplification, only with detectable, about 50 μ l test plasma or saliva sample need to be added detection hole In the middle of the respective reaction hole of plate, then detection orifice plate is put into holding structure disclosed in Application No. 201610658321.X and inclusion Detector (i.e. EFIRM instrument) in the detector of holding structure carries out electric field treatment and the detection of the signal of telecommunication.Detection process Using electric field action, accelerate reaction rate, shorten the response time, whole detection process can complete within half an hour, significantly contract Short detection time.
(2) high sensitivity and accuracy rate
The sensitivity of ARMS is about 1% about, if NGS adopts the sequencing of PCR amplicon or capture sequencing sensitivity big About in 1-5% about, sensitivity highest is digital pcr, about 0.1% about.The present invention adopts EFIRM technology for detection The sensitivity of ctDNA can reach 0.1%, suitable with digital pcr, reaches the advanced level of current ctDNA detection technique.
In terms of accuracy rate, heavy fragmentation (average length is only 166bp) is had due to ctDNA, content is low (accounts for periphery The ratio of blood dissociative DNA is less than 1%), the characteristic such as half-life short (about 2 hours), the therefore requirement to detection technique is high, at present In the ctDNA detectable of commercialization on market, product accuracy rate based on ARMS-PCR technology typically in 60-70%, Greatly differ from each other with clinical demand, thus seriously constrain its clinical practice.The Human epidermal growth factor receptor detection in Gene Mutation examination that the present invention provides Agent box, in clinical preliminary test, compares accuracy rate more than 95% with biopsy result, and the same period adopts the accurate of digital pcr technology Rate only has 70% about, and therefore, accuracy rate is substantially better than the product on existing market.
(3) with low cost
First, in terms of testing equipment, either ARMS-PCR or digital pcr all adopt fluorescence signal to detect at present, Testing equipment need to be equipped with the fluorescence detecting system of costliness, the quantitative real time PCR Instrument market price that therefore ARMS-PCR technology uses All about hundreds thousand of units, and digital pcr instrument more reaches more than 100 ten thousand yuan.The high-flux sequence instrument being used as NGS technology, no By be with illumina as representative optics sequencing or the quasiconductor sequencing system with ion proton as representative, market price is more Up to millions of units, fancy price seriously constrains it in clinical popularization and application.By comparison, the present invention is drawn using electric field The release led and e measurement technology, detection process utilizes electric field action, and reaction is quick, and final result detects as electronic signals, Therefore testing equipment is equipped with the fluorescence detecting system of costliness unlike ARMS-PCR and digital pcr, thus the cost of equipment is big Width reduces, and just corresponds to about the half of quantitative real time PCR Instrument, more not as good as 1/10th of high-flux sequence instrument.
Secondly, in terms of detectable, the principle based on nucleic acid hybridization for the EFIRM technology, using the electrochemistry of unique design Technology, the probe that detection is used need not carry out fluorescent labeling as ARMS-PCR, and due to saving DNA extraction and purification Step, therefore detectable cost is greatly reduced by comparison.Need through DNA extraction and purification, structure library, survey with NGS The preparation of sequence template, the several links of sequencing, are compared using multiple matched reagent boxes, the reagent cost of EFIRM has even more saved 90% More than, competitive advantage is obvious.
(4) requirement to clinical client hardware and personnel is greatly reduced
Due to DNA extraction and PCR amplification are not needed using EFIRM, thus do not deposit the problem that PCR easily pollutes, in clinic Clinic Gene Amplified laboratory need not be set up, operator also do not need to obtain the license on duty of Clinic Gene Amplified during use, Can be operated by those skilled in the art so that liquid Biopsy substantially reduces in the threshold of clinical application, beneficial to liquid Biopsy is promoted on a large scale, break away from existing ARMS-PCR, digital pcr, NGS technology can only minority satisfy the requirements large-scale The present situation that Grade A hospital is carried out.
Specific embodiment
Below in conjunction with embodiment, embodiment of the present invention is described in detail, but those skilled in the art will Understand, the following example is merely to illustrate the present invention, and be not construed as limiting the scope of the present invention.Unreceipted concrete in embodiment Condition person, the condition according to normal condition or manufacturer's suggestion is carried out.Agents useful for same or the unreceipted production firm person of instrument, are Can be by the commercially available conventional products bought and obtain.
Embodiment 1
It is respectively adopted following steps to different detection samples to detect：
1st, capture probe is fixed on the plate bottom of detection orifice plate
1.1pyrrole and the Compound mixed solution of capture probe (abbreviation CP)：
Take 1 1.5ml centrifuge tube, sequentially add ultra-pure water 885 μ l, 100 μ l 3M KCl, the concussion that is vortexed mixes, centrifugation； Add 5 μ l pyrrole, the concussion that is vortexed mixes, centrifugation；Add 100 μM of capture probe 10 μ l；It is vortexed after concussion mixes and be centrifuged, standby With.
1.2 sample-adding：
On the detection orifice plate in 96 holes, each hole adds the mixed liquor having prepared of 30 μ l, and during sample-adding, pipette tips press close to hole Bottom, but be not exposed to bottom electrode, add rear-inclined or to pat the detection orifice plate electrode surface that makes liquid in hole equal Even covering, then carries out electric field operation at once on EFIRM detector.
1.3EFIRM electric field treatment：
The respective column tested is selected on EFIRM software, electric pulse field parameter is set to：Voltage A：350mV, 1s；Voltage B:950mV, 1s；Carry out 5 circulations.Electric field treatment finishes, and takes out at once, cleaning detection orifice plate.
1.4 detection orifice plate cleanings：
Select on washing trigger program corresponding experiment row, cleaning procedure select (2bottom, 2top), washing liquid select 2 × SSC (0.05%SDS).Cleaning finishes, and carries out the operation of next step sample loading at once.
2nd, sample hybridization：
2.1 hybridization buffer pretreatment
Hybridization buffer (the hybridization buffer 1 using in content of the invention) 90 DEG C of water bath processing 10min in water-bath, Then room temperature places cooling 20min.
The preparation of 2.2 samples
Sample takes out from -20 DEG C of refrigerators, puts 4 DEG C of refrigerator defrostings into.After being completely dissolved, sample presses volume with hybridization buffer Ratio 1:2 mixing, are centrifuged after vortex oscillation, you can loading is detected.
2.3 sample-adding：
Detection orifice plate on, in corresponding hole add blank buffer, the negative control (WT) of respective concentration and Positive control (MT), applied sample amount 30 μ l.During sample-adding, pipette tips press close to the bottom in hole, but are not exposed to bottom electrode, add hypsokinesis Oblique or beating detection orifice plate makes electrode surface uniform fold in hole for the liquid, then carries out electric field operation at once on EFIRM.
2.4EFIRM electric field treatment：
The respective column tested is selected on EFIRM software, electric pulse field parameter is set to：Voltage A：300mV, 1s；Voltage B:500mV, 1s；Carry out 5 circulations.Electric field treatment finishes, and takes out at once, cleaning detection orifice plate.
2.5 detection orifice plate cleanings：
Select on washing trigger program corresponding experiment row, cleaning procedure select (2bottom, 2top), washing liquid select 2 × SSC (0.05%SDS).Cleaning finishes, and carries out DP sample-adding operation at once.
3rd, detection hybridization
3.1DP (detection probe) solution is prepared：
Take out, from 4 DEG C of refrigerators, the casein/PBS solution that caseic percetage by weight is 2%, take 1 1.5mL centrifugation Pipe, adds the casein/PBS of 990 μ l, adds 100 μM of DP 10 μ l, and the concussion that is vortexed mixes, and centrifugation is standby.
3.2 sample-adding：
Corresponding DP solution 30 μ l is added in corresponding hole according to experimental design, during sample-adding, pipette tips press close to the bottom in hole, but It is not exposed to bottom electrode, add rear-inclined or pat the electrode surface uniform fold that detection orifice plate makes liquid in hole, then Electric field operation is carried out at once on EFIRM.
3.3EFIRM electric field treatment：
The respective column tested is selected on EFIRM software, electric pulse field parameter is set to：Voltage A：300mV, 1s；Voltage B:500mV, 1s；Carry out 5 circulations.Electric field treatment finishes, and takes out at once, cleaning detection orifice plate.
3.4 detection orifice plate cleanings：
Select on washing trigger program corresponding experiment row, cleaning procedure select (2bottom, 2top), washing liquid select 2 × SSC (0.05%SDS).Cleaning finishes, and carries out sample-adding operation at once.
4、Reporter Hybridization
4.1Poly-HRP solution is prepared：
Take out, from 4 DEG C of refrigerators, the casein/PBS solution that caseic percetage by weight is 2%, take 1 1.5mL centrifugation Pipe, adds the casein/PBS (casein/PBS solution is put back to 4 DEG C of refrigerators) of 999 μ l, adds the poly-HRP of 1 μ l (to be purchased from Thermo fisher, name of product is PierceTMStreptavidin Poly-HRP, article No. is 21140, and unit specification is 0.5mL), the concussion that is vortexed mixes, and centrifugation is standby.
4.2 sample-adding：
Poly-HRP solution 30 μ l is added in corresponding hole according to experimental design, during sample-adding, pipette tips press close to the bottom in hole, but It is to be not exposed to bottom electrode, add rear-inclined or pat the electrode surface uniform fold that detection orifice plate makes liquid in hole, so After cover detection orifice plate lid, incubated at room 30min in laboratory table, timer is countdowned.Incubation time arrives, at once cleaning inspection Gaging hole plate.
4.3 detection orifice plate cleanings：
Select corresponding experiment row on washing trigger program, cleaning procedure selects (3bottom, 3top), and washing liquid selects PBST (0.1%Tween20).Cleaning finishes, and carries out TMB sample-adding operation at once.
5、Readout
5.1 sample-adding：
(purchased from thermo fisher, product article No. is to add TMB/H2O2 solution according to experimental design in corresponding hole 34022, entitled
Turbo tmb substrate solution), each hole adds 60 μ l, and during sample-adding, pipette tips press close to the bottom in hole, but are not exposed to Bottom electrode.Add and electric field operation is carried out at once on EFIRM.
5.2EFIRM electric field reading：
The respective column tested is selected on EFIRM software, electric pulse field parameter is set to：Voltage A：- 200mV, 60s, obtain To current indication.
In the present embodiment, different detection samples include negative group and positive group, and the gene that negative group measures is wild accordingly Raw type sequence, the gene that positive group measures is corresponding mutant sequences, and each sample arranges 20 parallel holes and detected.Phase Ying Di, every group of negative group (WT) and positive group (MT) using corresponding probe to detecting, to identify stablizing of its detection Property, the coefficient of variation (CV) result of the electric current of detection is as shown in table 2.
In table 2, coefficient of variation cv (coefficient of variation), also known as coefficient of dispersion (coefficient Of dispersion) or relative deviation (rsd), it is the ratio of standard deviation and meansigma methodss, is represented with percent, computing formula is： Cv=sd/mean × 100%.
Table 2 testing result
Wherein, the probe in table 2 and table 1 is to being corresponding in turn to.
From table 2 it can be seen that the present invention provide Human epidermal growth factor receptor gene mutation detection kit and using this test kit inspection Survey Human epidermal growth factor receptor gene mutation, the sample of the sequence content of as little as 1pM can be detected, detection sensitivity is high；The numerical value that detection obtains All below 15%, have is even as low as 3% about to mobility scale, detects good stability.Additionally, the feminine gender of detection variable concentrations Sample and positive sample, the numerical value that its detection obtains all has good linear relationship.
In addition, the wild-type sequence sample to series concentration gradient and mutant sequences sample, carry out 3 independent tests, 20 multiple holes of sample test every time, the numerical result interpretation that wild sample records is wild, for meeting, otherwise for not meeting；Prominent The numerical result interpretation that change sample records is then to meet into mutation, vice versa, concrete outcome is as shown in table 3.
Table 3 coincidence rate
In addition, the saliva sample to clinically have been acknowledged gene mutation type 190 for the method using the present embodiment (mutation 70, wild 120), 220 plasma samples (mutation 85, wild 135) and 200 hydrothorax samples are (prominent Become 90, wild 110) different probes is contrasted with Clinical detection result to carrying out double-blind, test result, inspection Survey the results are shown in Table 4.
Table 4 testing result
Sample Positive coincidence rate Negative match-rate
Blood plasma 98.8% 100%
Saliva 98.6% 100%
Hydrothorax 98.9% 100%
In addition, to the detection data of different samples as illustrated in tables 5-7.
The concrete detection case of table 5 saliva sample
The concrete detection case of table 6 plasma sample
The concrete detection case of table 7 hydrothorax sample
Wherein, the computing formula of the positive coincidence rate in table 4 and negative match-rate is as shown in table 8.
Table 8 sample statistics
Positive coincidence rate=a/ (a+c) × 100%；
Negative match-rate=b/ (b+d) × 100%.
Can be seen that whether plasma sample or saliva sample or hydrothorax sample standard deviation from table 4-7 has preferably Clinical detection coincidence rate, accuracy rate is high, more than more than 95%, meets the demand of Clinical detection application.
When said method detects to negative sample and positive sample, detect that the current value difference obtaining is big, can be very well The different samples of differentiation.As to the negative sample of saliva using 34 probes to detecting, the current value that obtains 41.6～ 65.6 (- nA), and the current value of positive sample is in 88.4～141.4 (- nA)；Correspondingly, to same plasma sample, negative sample Originally the current value obtaining is in 40.1～63.7 (- nA), and the current value of positive sample is 83.6～139.2.
In addition, the concentration of each component in change test kit, detected, result is basically identical with the above results.
In summary, the present invention provide Human epidermal growth factor receptor gene mutation detection kit and using this test kit detect people EGFR genetic mutation, easy and simple to handle, sample noinvasive, and required sample is few, and whole detection process can complete within half an hour； There is susceptiveness good, 0.1% can be reached, accuracy rate is high, more than more than 95%；Low cost, compared with NGS, cost savings 90% More than；Easily operated, operator are required low.
Embodiment 2
Sample to be detected is detected, comprises the following steps that：
1st, capture probe is fixed on the plate bottom of detection orifice plate
1.1pyrrole and the Compound mixed solution of capture probe (abbreviation CP)：
Capture solution contains following component：The percetage by weight of thiophene is the concentration of 5%, NaCl is 2mol/L, and capture is visited The concentration of pin is 1.5 μm of ol/L.
1.2 sample-adding：
On the detection orifice plate in 96 holes, each hole adds the pyrrole having prepared and the CP mixed liquor of 20 μ l, during sample-adding Pipette tips press close to the bottom in hole, but are not exposed to bottom electrode, add rear-inclined or beating detection orifice plate makes liquid in hole Electrode surface uniform fold, then carries out electric field operation at once on EFIRM detector.
1.3EFIRM electric field treatment：
The respective column tested is selected on EFIRM software, electric pulse field parameter is set to：Voltage A：500mV, 1s；Voltage B:1500mV, 1s；Carry out 10 circulations.Electric field treatment finishes, and takes out at once, cleaning detection orifice plate.
1.4 detection orifice plate cleanings：
Select corresponding experiment row on washing trigger program, cleaning procedure selects (2bottom, 2top), and washing liquid selects 2XSSC (0.06%SDS).Cleaning finishes, and carries out the operation of next step sample loading at once.
2nd, sample hybridization：
2.1 hybridization buffer pretreatment
Hybridization buffer (the hybridization buffer 3 using in content of the invention) 95 DEG C of water bath processing 5min in water-bath, so Room temperature places cooling afterwards.
The preparation of 2.2 samples
Sample takes out from -20 DEG C of refrigerators, puts 4 DEG C of refrigerator defrostings into.After being completely dissolved, sample presses volume with hybridization buffer Ratio 1:2.5 mixing, are centrifuged after vortex oscillation, you can loading is detected.
2.3 sample-adding：
Detection orifice plate on, in corresponding hole add blank buffer, the negative control (WT) of respective concentration and Positive control (MT), applied sample amount 80 μ l.During sample-adding, pipette tips press close to the bottom in hole, but are not exposed to bottom electrode, add hypsokinesis Oblique or beating detection orifice plate makes electrode surface uniform fold in hole for the liquid, then carries out electric field operation at once on EFIRM.
2.4EFIRM electric field treatment：
The respective column tested is selected on EFIRM software, electric pulse field parameter is set to：Voltage A：500mV, 1s；Voltage B:800mV, 1s；Carry out 10 circulations.Electric field treatment finishes, and takes out at once, cleaning detection orifice plate.
2.5 detection orifice plate cleanings：
Select corresponding experiment row on washing trigger program, cleaning procedure selects (2bottom, 2top), and washing liquid selects 2XSSC (0.06%SDS).Cleaning finishes, and carries out DP sample-adding operation at once.
3rd, detection hybridization
3.1DP (detection probe) solution is prepared：
Detection solution is using PBS as solvent, and wherein caseic percetage by weight is 5%, and the concentration of detection probe is 1.5 μm of ol/L, the concussion that is vortexed mixes, and centrifugation is standby.
3.2 sample-adding：
Corresponding DP solution 20 μ l is added in corresponding hole according to experimental design, during sample-adding, pipette tips press close to the bottom in hole, but It is not exposed to bottom electrode, add rear-inclined or pat the electrode surface uniform fold that detection orifice plate makes liquid in hole, then Electric field operation is carried out at once on EFIRM.
3.3EFIRM electric field treatment：
The respective column tested is selected on EFIRM software, electric pulse field parameter is set to：Voltage A：500mV, 1s；Voltage B:800mV, 1s；Carry out 8 circulations.Electric field treatment finishes, and takes out at once, cleaning detection orifice plate.
3.4 detection orifice plate cleanings：
Select corresponding experiment row on washing trigger program, cleaning procedure selects (2bottom, 2top), and washing liquid selects 2XSSC (0.06%SDS).Cleaning finishes, and carries out sample-adding operation at once.
4、Reporter Hybridization
4.1Poly-HRP solution is prepared：
Using PBS as solvent, wherein caseic percetage by weight is the percentage by volume of 5%, poly-HRP is 0.2% (purchased from thermo fisher, name of product is PierceTMStreptavidin Poly-HRP, article No. is 21140, and unit is advised Lattice are 0.5mL), the concussion that is vortexed mixes, and centrifugation is standby.
4.2 sample-adding：
Poly-HRP solution 20 μ l is added in corresponding hole according to experimental design, during sample-adding, pipette tips press close to the bottom in hole, but It is to be not exposed to bottom electrode, add rear-inclined or pat the electrode surface uniform fold that detection orifice plate makes liquid in hole, so After cover detection orifice plate lid, incubated at room 40min in laboratory table, timer is countdowned.Incubation time arrives, at once cleaning inspection Gaging hole plate.
4.3 detection orifice plate cleanings：
Select corresponding experiment row on washing trigger program, cleaning procedure selects (3bottom, 3top), and washing liquid selects PBST (0.15%Tween20).Cleaning finishes, and carries out TMB sample-adding operation at once.
5、Readout
5.1 sample-adding：
(purchased from thermo fisher, product article No. is to add TMB/H2O2 solution according to experimental design in corresponding hole 34022, entitled
Turbo tmb substrate solution), each hole adds 80 μ l, and during sample-adding, pipette tips press close to the bottom in hole, but are not exposed to Bottom electrode.Add and electric field operation is carried out at once on EFIRM.
5.2EFIRM electric field reading：
The respective column tested is selected on EFIRM software, electric pulse field parameter is set to：Voltage A：- 300mV, 100s, obtain To current indication.
Embodiment 3
Sample to be detected is detected, comprises the following steps that：
1st, capture probe is fixed on the plate bottom of detection orifice plate
1.1pyrrole and the Compound mixed solution of capture probe (abbreviation CP)：
Capture solution contains following component：The percetage by weight of aniline is the concentration of 0.1%, NaCl is 0.01mol/L, catches The concentration obtaining probe is 0.5 μm of ol/L.
1.2 sample-adding：
On the detection orifice plate in 96 holes, each hole adds the pyrrole having prepared and the CP mixed liquor of 80 μ l, during sample-adding Pipette tips press close to the bottom in hole, but are not exposed to bottom electrode, add rear-inclined or beating detection orifice plate makes liquid in hole Electrode surface uniform fold, then carries out electric field operation at once on EFIRM detector.
1.3EFIRM electric field treatment：
The respective column tested is selected on EFIRM software, electric pulse field parameter is set to：Voltage A：200mV, 5s；Voltage B:800mV, 5s；Carry out 3 circulations.Electric field treatment finishes, and takes out at once, cleaning detection orifice plate.
1.4 detection orifice plate cleanings：
Select corresponding experiment row on washing trigger program, cleaning procedure selects (2bottom, 2top), and washing liquid selects 2XSSC (0.04%SDS).Cleaning finishes, and carries out the operation of next step sample loading at once.
2nd, sample hybridization：
2.1 hybridization buffer pretreatment
Hybridization buffer (the hybridization buffer 6 using in content of the invention) 85 DEG C of water bath processing 15min in water-bath, Then room temperature places cooling.
The preparation of 2.2 samples
Sample takes out from -20 DEG C of refrigerators, puts 4 DEG C of refrigerator defrostings into.After being completely dissolved, sample presses volume with hybridization buffer Ratio 1:1.5 mixing, are centrifuged after vortex oscillation, you can loading is detected.
2.3 sample-adding：
Detection orifice plate on, in corresponding hole add blank buffer, the negative control (WT) of respective concentration and Positive control (MT), applied sample amount 20 μ l.During sample-adding, pipette tips press close to the bottom in hole, but are not exposed to bottom electrode, add hypsokinesis Oblique or beating detection orifice plate makes electrode surface uniform fold in hole for the liquid, then carries out electric field operation at once on EFIRM.
2.4EFIRM electric field treatment：
The respective column tested is selected on EFIRM software, electric pulse field parameter is set to：Voltage A：200mV, 5s；Voltage B:300mV, 5s；Carry out 3 circulations.Electric field treatment finishes, and takes out at once, cleaning detection orifice plate.
2.5 detection orifice plate cleanings：
Select corresponding experiment row on washing trigger program, cleaning procedure selects (2bottom, 2top), and washing liquid selects 2XSSC (0.04%SDS).Cleaning finishes, and carries out DP sample-adding operation at once.
3rd, detection hybridization
3.1DP (detection probe) solution is prepared：
Detection solution is using PBS as solvent, and wherein caseic percetage by weight is 0.1%, the concentration of detection probe For 0.5 μm of ol/L, the concussion that is vortexed mixes, and is centrifuged, standby.
3.2 sample-adding：
Corresponding DP solution 30 μ l is added in corresponding hole according to experimental design, during sample-adding, pipette tips press close to the bottom in hole, but It is not exposed to bottom electrode, add rear-inclined or pat the electrode surface uniform fold that detection orifice plate makes liquid in hole, then Electric field operation is carried out at once on EFIRM.
3.3EFIRM electric field treatment：
The respective column tested is selected on EFIRM software, electric pulse field parameter is set to：Voltage A：200mV, 5s；Voltage B:300mV, 5s；Carry out 3 circulations.Electric field treatment finishes, and takes out at once, cleaning detection orifice plate.
3.4 detection orifice plate cleanings：
Select corresponding experiment row on washing trigger program, cleaning procedure selects (2bottom, 2top), and washing liquid selects 2XSSC (0.04%SDS).Cleaning finishes, and carries out sample-adding operation at once.
4、Reporter Hybridization
4.1Poly-HRP solution is prepared：
Using PBS as solvent, wherein caseic percetage by weight is that the percentage by volume of 0.1%, poly-HRP is 0.05% (purchased from thermo fisher, name of product is PierceTMStreptavidin Poly-HRP, article No. is 21140, Unit specification is 0.5mL), the concussion that is vortexed mixes, and centrifugation is standby.
4.2 sample-adding：
Poly-HRP solution 20-80 μ l is added in corresponding hole according to experimental design, during sample-adding, pipette tips press close to the bottom in hole, But it is not exposed to bottom electrode, add rear-inclined or pat the electrode surface uniform fold that detection orifice plate makes liquid in hole, Then detection orifice plate lid, incubated at room 20-40min in laboratory table are covered, timer is countdowned.Incubation time arrives, at once Cleaning detection orifice plate.
4.3 detection orifice plate cleanings：
Select corresponding experiment row on washing trigger program, cleaning procedure selects (3bottom, 3top), and washing liquid selects PBST (0.05%Tween20).Cleaning finishes, and carries out TMB sample-adding operation at once.
5、Readout
5.1 sample-adding：
(purchased from thermo fisher, product article No. is to add TMB/H2O2 solution according to experimental design in corresponding hole 34022, entitled
Turbo tmb substrate solution), each hole adds 30 μ l, and during sample-adding, pipette tips press close to the bottom in hole, but are not exposed to Bottom electrode.Add and electric field operation is carried out at once on EFIRM.
5.2EFIRM electric field reading：
The respective column tested is selected on EFIRM software, electric pulse field parameter is set to：Voltage A：- 100mV, 40s, obtain To current indication.
Similarly, detected by the way of the method for the embodiment of the present invention 2 and 3 offer is same as Example 1, obtained Result basically identical with embodiment 1 result.
It can be seen that, the present invention provide Human epidermal growth factor receptor gene mutation detection kit and using this test kit detect Human epidermal growth factor receptor base Because of mutation, easy and simple to handle, sample noinvasive, and required sample is few, and whole detection process can complete within half an hour；There is spirit Quick property is good, can reach 0.1%, and accuracy rate is high, more than more than 95%；Low cost, compared with NGS, cost savings more than 90%；Easily In operation, operator are required low.
Although illustrate and describing the present invention with specific embodiment, but it will be appreciated that without departing substantially from the present invention's Many other changes can be made in the case of spirit and scope and change.It is, therefore, intended that in the following claims Including all such changes and modifications belonging in the scope of the invention.

Claims (10)

1. it is used for the probe of Human epidermal growth factor receptor detection in Gene Mutation it is characterised in that described probe includes the arbitrary of 1-35 probe centering Plant or multiple, each probe centering includes capture probe and detection probe；
The nucleotide sequence of 1-35 probe pair such as SEQ ID NO：Shown in 1-70.
2. probe according to claim 1 is it is characterised in that be provided with label in described detection probe, described labelling Thing is preferably digoxin, biotin, any one in marked by fluorescein isothiocyanate thing.
3. Human epidermal growth factor receptor gene mutation detection kit it is characterised in that at least the probe containing claim 2, enzyme and with institute State any one or more in the corresponding substrate of enzyme；
The material being combined with the label of setting in described detection probe is marked with described enzyme；
Preferably, when the label in detection probe is digoxin or Fluorescein isothiocyanate, on enzyme, the material of labelling is corresponding For DigiTAb or Fluorescein isothiocyanate antibody；When the label in detection probe is biotin, labelling on enzyme Material is Avidin；
Preferably, described enzyme is horseradish peroxidase polymer or alkali phosphatase；
When described enzyme is horseradish peroxidase polymer, substrate is TMB and H2O2Mixture, o-phenylenediamine, ABTS, 3- Any one in (4- hydroxyl)；
When described enzyme is alkali phosphatase, substrate is para-nitro-pheneye phosphate, the compositionss of BCIP and NBT, 4- Nitrobenzol phosphorus Any one in acid disodium, naphthols AS-BI phosphate, naphthols-AS-MX- phosphate.
4. Human epidermal growth factor receptor gene mutation detection kit according to claim 3 is it is characterised in that described test kit also includes For the fixture that described capture probe is fixing, hybridization buffer, closed protein, PBS, cleanout fluid, detection orifice plate；
Described closed protein is preferably casein, hyclone, any one in calf serum.
5. Human epidermal growth factor receptor gene mutation detection kit according to claim 4 is it is characterised in that described fixture includes leading Electric polymer material and salts compound；
Described conducting polymer materials are any one in aniline, thiophene and pyrroles's electroconductive molecule monomer；
Described salts compound is chlorate, nitrate, any one in sulfate；
Preferably, presented in capturing solution, described capture solution contains following for described capture probe and described fixture Composition：The percentage by volume of conducting polymer materials is 0.1%～5%, the concentration of salts compound be 0.01mol/L～ 2mol/L, the concentration of capture probe is 0.5-1.5 μm of ol/L.
6. Human epidermal growth factor receptor gene mutation detection kit according to claim 5 is it is characterised in that described capture probe and institute State fixture and be attached to described detection orifice plate presence；
Specifically adopt following methods preparation：Described capture solution is added in the reacting hole of described detection orifice plate, then in electricity Field processes and carries out polyreaction, cleans.
7. Human epidermal growth factor receptor gene mutation detection kit according to claim 3 is it is characterised in that described detection probe is to examine Presented in surveying solution；
Described detection solution is that the wherein percetage by weight of closed protein is 0.1%～5%, detection probe using PBS as solvent Concentration be 0.5-1.5 μm of ol/L.
8. Human epidermal growth factor receptor gene mutation detection kit according to claim 3 is it is characterised in that described enzyme is with enzyme liquid Form exists, and described enzyme liquid is that the wherein percetage by weight of closed protein is 0.1%～5%, the body of enzyme using PBS as solvent Long-pending percent is 0.05%～0.2%.
9. Human epidermal growth factor receptor gene mutation detection kit according to claim 4 is it is characterised in that described cleanout fluid includes containing There is 0.1 × SSC-2 × SSC mixed liquor that mass concentration is 0.04%-0.06%SDS and contain percentage by volume for 0.05%- The PBS solution of 0.15%Tween20.
10. a kind of method of detection Human epidermal growth factor receptor gene mutation is it is characterised in that comprise the following steps：
Capture solution described in claim 5 is added in detection orifice plate, electric field treatment 3-10 circulates, each circulates electric field Process program be：Voltage 200-500mV, 1-5s；Voltage 800-1500mV, 1-5s；
Electric field treatment is cleaned using described abluent after finishing, and is subsequently adding the mixed solution of sample to be tested and hybridization buffer, Electric field treatment 3-10 circulates, and the program of each circulation electric field treatment is：Voltage 200-500mV, 1-5s；Voltage 300-800mV, 1-5s；
Detection orifice plate cleaning, is subsequently adding the detection solution described in claim 7, electric field treatment 3-8 circulates, each circulation The program of electric field treatment is：Voltage 200-500mV, 1-5s；Voltage 300-800mV, 1-5s；
Detection orifice plate cleaning, is subsequently adding the enzyme liquid described in claim 8, cleans after incubation；
Add substrate, reading under the electric field treatment that voltage is -100～-300mV, obtain current value；
Described sample to be tested is body fluid, and described body fluid includes blood plasma, saliva, hydrothorax.
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