WO2018059576A1 - Probe for human egfr genetic mutation detection, kit and detection method thereof - Google Patents

Abstract

The invention provides a probe for human EGFR genetic mutation detection, a kit and a detection method thereof. The probe comprises one kind or several kinds of probes in 1 to 35 probe pairs. Each probe pair comprises a capture probe and a detection probe, and the nucleotide sequences of the 1 to 35 probe pairs are as shown in SEQ ID NO: 1 to 70. The invention also provides a kit for human EGFR genetic mutation detection.

Description

Probe, kit and detection method thereof for detecting human EGFR gene mutation

cross reference

The invention is entitled "Probes, kits and detection methods for detecting human EGFR gene mutations" filed on September 30, 2016, the invention patent application with the application number 201610873223.8, and December 26, 2016 Submitted, the utility model name is "detection electrode structure and detection orifice plate", the priority of the Chinese utility model patent application with the application number 201620769829.2, the entire contents of the above two Chinese patent applications, hereby based on all purposes, by reference The manner of the invention is incorporated in the present invention.

Technical field

The invention relates to the field of gene detection, in particular to a probe, a kit and a detection method thereof for detecting mutation of a human EGFR gene.

Background technique

Environmental factors such as air pollution have led to a sharp increase in the number of lung cancer patients in China in recent years. The incidence of lung cancer among 100,000 people increased from 39.6% in 2002 to 63% in 2011, especially in children and adolescents. In the past 30 years of reform and opening up, the number of deaths caused by lung cancer has increased by 465%. Lung cancer has become the number one malignant tumor in China in terms of morbidity and mortality. According to traditional histopathological classification, lung cancer can be divided into small cell lung cancer and non-small cell lung cancer. Non-small-cell Lung Cancer (NSCLC), including squamous cell carcinoma, adenocarcinoma, and large cell carcinoma, has slower cell growth and division and relatively late diffusion and metastasis compared with small cell lung cancer. Lung cancer accounts for about 80-85% of the total number of lung cancers.

The Epidermal Growth Factor Receptor (EGFR) is a receptor for epidermal growth factor (EGF) cell proliferation and signaling. EGFR belongs to the ErbB receptor family, which includes EGFR (ErbB-1), HER2/c-neu (ErbB-2), Her3 (ErbB-3) and Her 4 (ErbB-4). EGFR belongs to the tyrosine kinase type receptor. After EGFR binds to its ligand in the extracellular region, EGFR molecules form homodimers, and can also form heterodimers with other HER family molecules, followed by intracellular regions. The tyrosine kinase (TK) is activated, and the tyrosine residues are mutually phosphorylated, and then the downstream coupled proteins (PLCγ, aCBL, GRB2, SHC, p85) specifically bind to the phosphorylation site, transmitting signals downstream. The RAS-MAPK pathway, PI3K-AKT pathway, STAT pathway, etc., activate proliferation, apoptosis escape, angiogenesis, metastasis and other biological behaviors closely related to tumor development. In various types of tumors including lung cancer, overexpression of EGFR gene is frequently found, and it is also related to prognosis. Therefore, EGFR has attracted much attention as a molecular target.

Studies have shown that EGFR mutant genes are associated with the use of tyrosine kinase inhibitors (TKI), including Iressa (Iressa) and Tarceva (Troquet), and EGFR gene mutations affect TKI. Clinical efficacy. EGFR mutations mainly include four types: exon 19 deletion mutation, exon 2l point mutation, exon 18 point mutation and exon 20 insertion mutation. It has been found that more than 90% of the mutations in the EGFR gene are located in exons 19 and 21, and the most common EGFR mutations are the exon 19LREA deletion and the exon 21L858R mutation, both of which result in activation of the tyrosine kinase domain, and All of them are sensitive mutations in TKI. The T790M mutation in exon 20 is associated with acquired resistance to EGFR-TKI, and the clinical significance of many types of mutations is still unclear. The positive rate of EGFR gene-sensitive mutations in lung adenocarcinoma patients is about 10% in the Caucasian population, and about 50% in the Asian population and in China.

At present, the detection of EGFR gene mutation is mainly divided into the detection of tumor tissue samples and the detection of circulating tumor DNA (ctDNA). At present, tumor tissue samples obtained by biopsy or surgery are mainly used to detect EGFR gene mutations, but invasive methods are required for obtaining tumor tissue specimens, which often increases patient suffering and creates additional surgical risks. And the tumor is heterogeneous. For a cancer patient who has already metastasized, only taking a certain part of the cancer tissue by puncture or surgery does not reflect the overall condition of the patient. Second, some patients' own conditions determine that they are not suitable for tissue biopsy, and some tumors have the risk of accelerating metastasis after being disturbed by puncture or surgery. Finally, the organization of biopsy has many problems such as high cost and long waiting time, and its hysteresis is also unfavorable for the treatment of patients.

Therefore, in recent years, the concept of "liquid biopsy" is emerging. The basic idea is to use blood samples such as blood to replace tumor tissue samples for pathology and molecular biology, and to detect tumor circulation in body fluid samples (mainly blood). DNA has become a trend to obtain information on tumor gene mutations. Compared with current standard tissue biopsy, revolutionary liquid biopsy has the following irreplaceable advantages: small trauma, reproducibility, homogenization heterogeneity, real-time judgment of efficacy, and dynamic adjustment of treatment decisions as the tumor progresses. Therefore, the 2015 MIT Technology Review released the annual breakthrough technology (Breakthrough Technologies 2015), ASCO annual progress (Clinical In the cancer advance 2015), liquid biopsy is on the list for the next decade. By detecting ctDNA to track specific gene changes in tumors throughout the course of the disease, it is of great value for tumor screening, diagnosis, efficacy monitoring and prognosis judgment. It can also explore the molecular mechanism of tumor metastasis, recurrence and drug resistance, and identify new targets. To the treatment site, etc., the detection of ctDNA has become one of the three hot trends in tumor liquid biopsy applications (note: the other two are CTC and exosomes).

At present, the main methods for detecting gene mutations include direct sequencing (PCR-sanger sequencing), Amplification refractory mutation system (ARMS), and Denaturing high-performance liquid chromatograph (DHPLC). High resolution melting (HRM), allele-specific Taqman polymerase chain reaction (CAST-PCR), digital PCR (dPCR), High-throughput sequencing (HTS) and the like.

1. Direct sequencing method. Direct sequencing method firstly designed PCR primers for the mutation site, and obtained the target gene fragment by PCR amplification. Then the sanger sequencing of the PCR product was carried out and the sequencing results were analyzed. After the preliminary analysis is completed, the PCR product of the sample which may carry the mutation information needs to be recovered by a method such as gelatinization, and the recovered PCR product is ligated to the cloning vector (generally a plasmid), and the positive clone is selected for sequencing to confirm the mutation.

2. Amplify the repression mutation system. The method utilizes the principle that the 3'-end base of the PCR primer must be complementary to the template DNA for efficient amplification, and the specific PCR amplification primer for the mutation site is designed. Under strict amplification conditions, only the primer 3' When the base is paired with the template, the PCR reaction can proceed normally, thereby detecting the mutation. By designing two upstream primers, one of the 3' ends is complementary to the normal gene sequence, and the other 3' end is complementary to the mutated gene sequence. Two upstream primers and a common downstream primer are added for detection, and two parallel PCR, based on the results of two PCR reactions, can distinguish whether there is a mutation and whether the mutation is homozygous or heterozygous. The detection sensitivity of the ARMS method is about 1%.

3. Denaturing high performance liquid chromatography. The principle of this method is based on the difference in the melting characteristics of the mismatched heteroduplex DNA and the perfectly matched homozygous double-stranded DNA, and separation by chromatographic methods. Due to the mismatch of the heteroduplex at the mutation site, it is easy to form a "Y" structure, and the ability to bind to the stationary phase of the column is reduced. Therefore, the hybrid double-stranded DNA preferentially elutes than the homozygous double-stranded DNA. A change in the elution peak can determine whether or not there is a mutation. The method has the advantages that both known and unknown mutations can be detected, but the target gene fragment needs to be obtained by PCR before the detection, and the reaction process needs to open the reaction tube, which is easy to cause pollution, the detection sensitivity is about 5%, and for the positive result. It is not possible to determine the specific type of mutation, and ultimately requires sequencing verification.

4. High resolution dissolution curve. The method is based on the physical properties of the nucleic acid fragment, and the gene mutation is analyzed by monitoring the dissolution curve of the PCR product by binding the saturated dye to the PCR amplification product. The detection sensitivity of this method is about 5%. For the positive result, the specific mutation type cannot be determined, and finally the sequencing verification is needed.

5. Allele-specific Taqman polymerase chain reaction. The method uses an optimized Taqman probe to block the binding of PCR primers to wild-type DNA by a specific MGB probe, so that the mutant DNA can be preferentially amplified, and the sensitivity of the detection method is about 1%.

6, digital PCR. Digital PCR is a method of absolute quantification based on single-molecule PCR methods for counting nucleic acids. The microfluidic or microtiter method is mainly used to disperse a large amount of the diluted nucleic acid solution into the microreactor or droplet of the chip, and the number of nucleic acid templates per reactor is less than or equal to one. Thus, after the PCR cycle, the reactor with one template of the nucleic acid molecule gives a fluorescent signal, and the reactor without the template has no fluorescent signal. Based on the relative ratio and the volume of the reactor, the nucleic acid concentration of the original solution can be derived. The detection sensitivity of digital PCR is about 0.1%.

7. High-throughput sequencing technology. This technology is a revolutionary change to the traditional Sanger sequencing (called a generation of sequencing technology), the basic principle is to synthesize while sequencing, based on Sanger and other sequencing methods, using different colors of fluorescence to mark four different dNTPs, when When DNA polymerase synthesizes a complementary strand, each additional dNTP releases a different fluorescence, capturing H+ changes during DNA synthesis based on captured fluorescent signals (such as the Illumina sequencing platform) or through a semiconductor sequencing chip (eg, Life ion) The torrent sequencing platform) is processed by a specific computer software to obtain sequence information of the DNA to be tested, and sequence determination of hundreds of thousands to millions of nucleic acid molecules at a time, also known as next generation sequencing , NGS). The biggest advantage of this technology is its high throughput, which can simultaneously sequence multiple samples in parallel. In addition to known mutations, unknown mutations can be found, and the detection sensitivity is about 1-5%. The detection process of high-throughput sequencing generally includes DNA extraction and purification, construction of libraries, sequencing template preparation, sequencing, and biological information analysis.

However, the prior art has the following drawbacks:

Direct sequencing. The direct sequencing method has the advantage of being able to determine the range and type of mutation. However, due to the low sanger sequencing flux and the limitation of the sequencing method itself, the sensitivity is not high, and only the gene mutation with the content greater than 20% can be detected, which is far from satisfactory. Practical application Need, especially for heterogeneous tumor somatic mutations, low sensitivity will result in a large number of missed tests. At the same time, the sequencing method has complicated detection operation and poor timeliness. The limitations of sequencing methods have been highlighted for the practical application of high timeliness and high sensitivity.

Amplification of the repressor mutation system requires real-time quantitative PCR detection technology, each pair of primers can only detect one mutation, and each mutation needs to establish a separate PCR reaction system, and it is cumbersome to detect multiple detection sites of multiple samples at the same time. Moreover, the method needs to extract purified DNA, and the requirement for the sample is large, and it is not suitable for detecting multiple gene mutation sites at the same time, and the PCR operation process is prone to aerosol pollution, which brings a risk of false positive. The allele-specific Taqman polymerase chain reaction also has the same disadvantages.

Denaturing high performance liquid chromatography and high resolution dissolution curve method and for the positive results can not determine the specific mutation type, and finally need to be verified by sequencing. The detection sensitivity of the two methods is about 5%, which cannot meet the needs of practical applications.

Although the digital PCR technology has high sensitivity, the technology is also very prone to false positive results. At the same time, due to its complicated operation, the requirements for experimental operation are extremely high. In addition, the high cost of equipment and reagents severely limits its large-scale application. High-throughput sequencing technology requires several steps such as DNA extraction and purification, library construction, sequencing template preparation, and sequencing. The operation is cumbersome and the requirements for experimental operation are extremely high. Similarly, the price of expensive equipment and reagents severely limits its large-scale application.

In view of this, the present invention has been specifically proposed.

Summary of the invention

One aspect of the present invention is to provide a probe set for detecting a mutation in a human EGFR gene, which consists of a capture probe and a detection probe that specifically captures a mutation site of a human EGFR gene, and a detection probe The side sequence of the mutation site is detected to achieve the purpose of accurate detection.

The contribution of the second aspect of the present invention is to provide a detection plate, a kit and a detection method for detecting human EGFR gene mutation based on EFIRM technology, and the kit requires only a small amount of sample detection, and the detection process utilizes an electric field to accelerate the reaction rate and shorten the reaction. Time, the entire inspection process can be completed in half an hour, greatly reducing the time required for testing, and high detection sensitivity and accuracy.

In order to achieve the above object of the present invention, the following technical solutions are adopted:

The invention provides a probe for detecting a mutation of a human EGFR gene, the probe comprising any one or more of a pair of 1-35 probes, each of which comprises a capture probe and a detection probe;

The nucleotide sequence of the 1-35 probe pair is as shown in SEQ ID NO: 1-70 of the Sequence Listing;

The probe provided by the present invention is designed in sequence according to the wild type sequence shown in SEQ ID NO: 71-91, 127-129 and the mutant sequence shown in SEQ ID NO: 92-126, wherein 1-35 probe Targeting to the mutant sequence shown in SEQ ID NOs: 92-126;

The wild type sequence set forth in SEQ ID NO: 71 corresponding to the mutant sequence set forth in SEQ ID NO: 92, 93, 94;

The mutant sequence set forth in SEQ ID NO: 96, 97, 99, 100, 101, 103, 111, 112, 113, 115 corresponds to the wild type sequence set forth in SEQ ID NO:73. The correspondence between all the mutant sequence numbers and the wild type sequences is shown in the table.

Applicants have undergone extensive testing to determine the sensitivity and specificity of capture probes and detection probes, and finally to screen for 1-35 probe pairs with good sensitivity and specificity. The 1-35 probe pair corresponds in turn to the above-described mutant sequence and wild type sequence.

The 1-3 probe pair in the probe for detecting human EGFR gene mutation provided by the present invention is designed for the exon 18 point mutation of EGFR, and the 4-28 probe pair is the exon 19 deletion mutation for EGFR. Designed, the 29-33 probe pair is designed for exon 20 mutations of EGFR, and the 34-35 probe pair is designed for exon 21 point mutations of EGFR. Specifically, the nucleotide sequence of the 1-3 probe pair is as shown in SEQ ID NO: 1-6; the nucleotide sequence of the capture probe in the probe pair is as shown in SEQ ID NO: 1, detecting The needle is shown in SEQ ID NO: 2; the nucleotide sequence of the capture probe in the 2 probe pair is shown in SEQ ID NO: 3, and the detection probe is shown in SEQ ID NO: 4. Accordingly, the remaining pairs of probes are analogous.

The capture probe obtained by the multiple screening of the invention cooperates with the detection probe, has high sensitivity and strong specificity, and can detect various samples containing the target sequence, and the result is accurate and reliable.

Specifically, the sequences involved in the present invention are shown in Table 1.

Table 1 sequence

The present invention uses the change of the electrical signal to realize the detection of the EGFR gene mutation site. Based on this, further, the detection probe is provided with a label, and the label is preferably digoxin, biotin, isothiocyanate. Any of the acid fluorescein labels. Thus, the detection probe binds to the subsequently added enzyme with the corresponding label to provide a basis for detecting changes in the electrical signal.

The invention is based on the above probe set disclosed by the invention, and provides a detection plate for detecting mutation of human EGFR gene based on EFIRM technology, wherein the bottom of the reaction hole is provided with an electrode for connecting the EFIRM detector. An electric field is applied to the solution in the reaction well, and the reaction wells are dispensed and fixed with capture probes in the probe combination, and each of the reaction wells is fixed with one of the capture probes.

Preferably, wherein the capture probe is mixed with the immobilizer, and is attached to the reaction well of the detection orifice by an electric field;

The fixture comprises a conductive polymer material and a salt ion compound;

The conductive polymer material is selected from at least one of aniline, thiophene and pyrrole conductive molecular monomers;

The salt ionic compound is at least one selected from the group consisting of a chloride salt, a nitrate salt, and a sulfate salt.

The present invention also provides a method for preparing the above detection orifice plate, wherein the capture probe is mixed with the immobilization material as a capture solution,

Adding the capture solution to the reaction well of the blank detection well plate, and the bottom of the reaction well is provided with an electrode for turning on the EFIRM detector to apply an electric field to the solution in the reaction well for polymerization;

After the EFIRM detector is turned on, an electric field is applied to the solution in the reaction well to carry out polymerization.

Preferably, the composition of the capture solution is as follows: the volume percentage of the conductive polymer material is 0.1% to 5%, the concentration of the salt ion compound is 0.01 mol/L to 2 mol/L, and the concentration of the capture probe is 0.5-1.5 μmol/ L. The conductive polymer material is selected from at least one of aniline, thiophene and pyrrole conductive molecular monomers;

The salt ionic compound is selected from at least one of a chloride salt, a nitrate salt, and a sulfate salt;

The parameters of the electric field are as follows: voltage A: 350 mV, 1 s; voltage B: 950 mV, 1 s; 5 cycles are performed.

The present invention also provides a human EGFR gene mutation detection kit, comprising at least one of the above probes, an enzyme, and a substrate corresponding to the enzyme;

The enzyme is labeled with a substance that binds to a label provided on the detection probe.

Preferably, when the label on the detection probe is digoxin or fluorescein isothiocyanate (abbreviated as FITC), the enzyme-labeled substance is correspondingly a digoxin antibody or a fluorescein isothiocyanate antibody; When the label on the detection probe is biotin, the enzyme-labeled substance is avidin.

That is, the detection probe and the enzyme can be bound by means of FITC and anti-FITC antibody or digoxin and digoxin antibody or avidin such as streptavidin and biotin.

Preferably, the enzyme is a horseradish peroxidase polymer (abbreviated Poly-HRP) or an alkaline phosphatase;

When the enzyme is a horseradish peroxidase polymer, the substrate is a mixture of TMB and H ₂ O ₂ , O-phenylenediamine (OPD), ABTS [2, 2'-azino-di- Any of (3-ethylbenziazobine sulfonate-6)], 3-(4-hydroxy);

Among them, the product after oxidation of OPD is dark orange or brown, and the product of ABTS reaction is blue-green.

When the enzyme is alkaline phosphatase, the substrate is a composition of p-nitropheny-phosphate disodium salt (pNPP), BCIP and NBT, disodium 4-nitrobenzene phosphate, naphthol Any of AS-BI phosphate, naphthol-AS-MX-phosphate.

Among them, pNPP produces a yellow water-soluble reaction product under the action of alkaline phosphatase; BCIP and NBT are one of the best chromogenic substrate combinations of alkaline phosphatase, the product is dark blue; disodium 4-nitrophenyl phosphate The product is yellow soluble; naphthol AS-BI phosphate, the product is red insoluble; naphthol-AS-MX-phosphate, the product is red insoluble.

The EGFR gene mutation detection kit provided by the invention provides convenient detection of EGFR gene mutation.

For easier detection, preferably, the kit further includes a fixture for immobilizing the capture probe, a hybridization buffer, a blocking protein, a PBS, a washing solution, and a detection well plate.

The specific structure of the detection orifice is shown in the name of the detection electrode and the detection orifice in the detection orifice as disclosed in the application No. 201620769829.2.

Further, the blocking protein is any one of casein, fetal bovine serum, and calf serum.

Each of the substances in the kit provided by the present invention may be present alone or in the form of a mixed solution.

Further, the fixture comprises a conductive polymer material and a salt ion compound;

The conductive polymer material is any one of aniline, thiophene and pyrrole conductive molecular monomers;

The salt ionic compound is any one of a chloride salt, a nitrate salt, and a sulfate salt.

The salt may be sodium, potassium, magnesium, ammonium or the like; for example, the chloride salt may be sodium chloride, potassium chloride, magnesium chloride, ammonium chloride, etc. Similarly, the nitrate may be sodium nitrate, potassium nitrate or nitric acid. Magnesium, ammonium nitrate, etc., the sulfate may be sodium sulfate, potassium sulfate, magnesium sulfate, ammonium sulfate or the like.

When the EGFR gene mutation detection kit provided by the invention is used for detection, the first step is to fix the capture probe on the detection well plate by polymerization under electric field conditions, which provides a basis for subsequent detection.

Therefore, in order to facilitate the detection operation, preferably, the capture probe and the immobilizer are present in the form of a capture solution containing the following components: a volume percentage of the conductive polymer material of 0.1% to 5%, salt The concentration of the ionic compound is from 0.01 mol/L to 2 mol/L, and the concentration of the capture probe is from 0.5 to 1.5 μmol/L.

That is, when the detection is performed, the capture solution can be directly added to the reaction well of the detection well plate. Generally, in the 96-well detection well plate, the volume of each reaction well is about 300 μl, and 20-80 μl of the capture solution is added. All right.

After the capture solution is added to the reaction well of the detection well plate, electric field treatment is performed, so that the components in the capture solution are fixed in the reaction well of the detection well plate to facilitate the subsequent reaction.

In order to improve the convenience of detection, further, the capture probe and the fixture are attached to the detection orifice plate;

Specifically, it is prepared by adding the capture solution to the reaction well of the detection well plate, and then washing it after the electric field treatment.

When cleaning, the procedure is: cleaning procedure from the bottom: aspirating while injecting liquid; cleaning procedure from top to bottom: first injecting liquid, then aspirating; repeating the above steps.

After washing, a mixture of the sample to be tested and the hybrid buffer is added, wherein the hybrid buffer is treated in a water bath at 85-95 ° C for 5-15 min, then left to cool to room temperature, and mixed with the sample to be tested at a volume ratio of 1:1.5-2.5. Then, 20-80 μl was added to the reaction well of the detection well plate, subjected to electric field treatment, and then washed by the same washing step as above.

The hybridization buffer of the present invention provides a good environment for hybridization of the probe, and any hybridization buffer that achieves this function is within the scope of the present invention. The hybridization buffer as used may be any one of the following;

Hybridization buffer 1: containing Ficoll 400 1g / L, PVP360 1g / L, BSA 1g / L, NaH 2 PO 4 80mM, Na 2 HPO 4 420mM, EDTA 2.5mM, SDS 6g / L, NaCl 43.8g / L, dihydrate Sodium citrate 36.9 g/L, pH 7-8; the buffer was heated to 65 °C prior to use.

Hybrid buffer 2: in this solution, the volume percentage of 20×SSC is 10%, the volume percentage of 100×Denhardt is 5%, the mass concentration of Denatured salmon DNA is 0.1 mg/ml, and the volume percentage of formamide is 40%;

Hybrid buffer 3: 50% formamide, 2 x SSC, 10% dextran sulfate (pH 7);

Hybrid buffer 4: 1.5 M NaCl, 40 mM sodium phosphate buffer pH 7.2, 10 mM EDTA pH 8, 10 x Denhardt's solution, 0.2% SDS;

Hybrid buffer 5: 20-100 nM Tris-HCl, 20-200 nM MgCl ₂ , 0.01%-5% dextran sulfate;

Hybrid buffer 6: 5 × SSC, 1% blocking protein, 0.1% N-lauroyl sarcosine, 0.02% SDS;

Hybrid buffer 7: 0.5 M sodium dihydrogen phosphate, pH 7.5; 10 mM EDTA, 7% SDS.

The percentages in the above hybrid buffer refer to the mass percentage, and the content of each component is also the final concentration of each component in the whole solution.

Further, the content of each component in the above-described hybrid buffer of the present invention may vary slightly, and such variation is also within the scope of the present invention.

Further, the detection probe is present in the form of a detection solution;

The detection solution is PBS as a solvent, wherein the weight percentage of the blocking protein is 0.1% to 5%, and the concentration of the detection probe is 0.5 to 1.5 μmol/L.

The role of blocking proteins such as casein in the detection solution is to block non-specific sites in the assay to increase the sensitivity and accuracy of the assay.

Further, 20-80 μl of the detection solution was added to the reaction well of the detection well plate, subjected to electric field treatment, and then washed using the same washing step as described above.

Further, the enzyme is in the form of an enzyme solution;

The enzyme solution is PBS as a solvent, wherein the weight percentage of the blocking protein is 0.1% to 5%, and the volume percentage of the enzyme is 0.05% to 0.2%.

In order to block non-specific sites in the assay and increase the sensitivity and accuracy of the assay, the enzyme solution also contains a blocking protein. Correspondingly, the enzyme solution is added in an amount of 20-80 μl, incubated at room temperature for 20-40 min, and the well plate is washed for cleaning, and the washing procedure is repeated one more time than the above washing procedure.

Further, the cleaning solution comprises a 0.1×SSC-2×SSC mixture containing a mass concentration of 0.04% to 0.06% SDS and a PBS solution containing a volume percentage of 0.05% to 0.15% Tween20.

PBST (0.1% Tween 20) 1 L: 949 ml of double distilled water was added to 50 ml of 20×PBS, mixed, and 1 ml of Tween 20 was added and mixed.

In the present invention, two kinds of cleaning liquids are used, and the steps before adding the enzyme liquid are all washed with a 0.1×SSC-2×SSC mixture having a mass concentration of 0.04%-0.06% SDS, and washed with PBST after adding the enzyme.

By cleaning, non-specific binding and other unreacted components are removed to improve the accuracy of the detection.

Further, the above-mentioned mixed liquid of the present invention is the concentration of the working liquid. However, depending on the demand, the mother liquid may be first prepared in accordance with the concentration of the above working liquid, and may be appropriately diluted at the time of use. Therefore, a corresponding concentration of the mother liquor is also within the scope of the present invention.

Among them, 20×PBS 1L formula is: potassium dihydrogen phosphate: 5.4g, disodium hydrogen phosphate: 28.4g, sodium chloride: 160g, potassium chloride 4g, Add about 800 mL of deionized water to dissolve thoroughly, then add concentrated hydrochloric acid to adjust the pH to 7.4, and finally make up to 1 L. Correspondingly, PBS is 1×PBS, and 20×PBS can be diluted 20 times.

The cleaning solution is also generally configured with a high concentration of SSC and SDS, and then the corresponding dilution can be performed.

Compared with the prior art, the human EGFR gene mutation detection kit provided by the invention has the advantages of being precise, reliable, fast, convenient and economical, and is advantageous for repeated sampling and detection, and has wide application range, and the sampling is harmless to the human body. The ideal liquid biopsy product.

The invention also provides a method for detecting a mutation in a human EGFR gene, comprising the steps of:

(1) using the above detection orifice plate, or

The capture probe comprising the probe set according to claim 1 or 2 is added to a blank detection well plate, and the bottom of the reaction well is provided with an electrode for applying the solution in the reaction well after the EFIRM detector is turned on. The electric field is subjected to a polymerization reaction; after the EFIRM detector is turned on, a first electric field is applied to the solution in the reaction well to carry out a polymerization reaction; after the electric field treatment is completed, the detection orifice plate is cleaned;

The parameters of the first electric field processing are: voltage 200-500 mV, 1-5 s; voltage 800-1500 mV, 1-5 s; 3-10 cycles;

(2) adding a mixed solution of the sample to be tested and the hybrid buffer, applying a second electric field to the reaction well: voltage 200-500 mV, 1-5 s; voltage 300-800 mV, 1-5 s; 3-10 cycles; cleaning detection orifice ;

(3) adding a detection solution, applying a third electric field: voltage 200-500 mV, 1-5 s; voltage 300-800 mV, 1-5 s; 3-8 cycles; cleaning the detection orifice;

The detection solution contains the detection probe in the probe set according to claim 1 or 2, and the concentration is 0.5-1.5 μmol/L;

(4) adding the enzyme solution, washing after incubation;

(5) Adding a substrate and reading under an electric field of a voltage of -100 to -300 mV, and obtaining a current value.

Preferably, the sample to be tested is a body fluid, and the body fluid includes plasma, saliva, and pleural effusion.

Preferably, the enzyme solution is PBS as a solvent, wherein the weight percentage of the blocking protein is 0.1% to 5%, and the volume percentage of the enzyme is 0.05% to 0.2%.

Preferably, the cleaning solution comprises a 0.1-2 x SSC mixture containing a mass concentration of 0.04% to 0.06% SDS and a PBS solution containing a volume percentage of 0.05% to 0.15% Tween20.

Preferably, the detection solution is PBS as a solvent, wherein the weight percentage of the blocking protein is 0.1% to 5%, and the concentration of the detection probe is 0.5-1.5 μmol/L.

The invention provides a method for detecting a mutation of a human EGFR gene, a capture solution is added to a detection well plate, a polymerization reaction occurs under the action of an electric field, and a capture probe is immobilized on the bottom of the plate; then a detection sample is added, and a capture probe captures a target gene mutation sample; The detection solution is added, and the biotin-labeled detection probe detects the sequence flanking the mutation site; the enzyme solution is added, the enzyme recognizes and binds to the biotin of the detection probe through the labeled avidin; and the substrate of the enzyme is added to generate oxidation. The reduction reaction generates a current, and the detection of the current signal completes the entire detection process.

The technical effects achieved by the technology of the present invention in a large number of embodiments have the following advantages compared with the prior art:

(1) Easy and fast operation

The prior art is based on PCR technology. First, DNA needs to be extracted and purified from the specimen, and then DNA amplification is required for PCR amplification. NGS is subjected to DNA extraction and purification, construction of a library, sequencing template preparation, and sequencing. The operation steps are complicated and the operation requirements are extremely high. Therefore, if ARMS-PCR and digital PCR technology are used in clinical practice, it takes 1-3 days to report. It takes 1-2 weeks for NGS technology to report. For tumor diseases that need to take appropriate treatment measures against time, this is the case. The reporting cycle severely limits its clinical application.

The kit provided by the invention detects exo-biomarkers by electric field induced release and measurement (EFIRM). For liquid biopsy, only a small amount of patient fluid sample (peripheral venous blood or a small amount of saliva collected by the patient) is needed, and the sample does not require DNA extraction. And amplification, only about 50 μl of the plasma or saliva sample to be tested and the detection reagent are added to the corresponding reaction wells of the detection well plate, and then the detection well plate is placed in the retention structure disclosed in application number 201610658321.X and includes The structure detector (ie EFIRM instrument) can perform electric field processing and electrical signal detection. The detection process utilizes the action of the electric field to speed up the reaction rate and shorten the reaction time. The entire detection process can be completed in half an hour, which greatly shortens the detection time.

(2) High sensitivity and accuracy

The sensitivity of ARMS is about 1%, and the sensitivity of NGS using PCR amplicon sequencing or capture sequencing is about 1-5% left. Right, the highest sensitivity is digital PCR, which is about 0.1%. The sensitivity of the invention for detecting ctDNA by using EFIRM technology can reach 0.1%, which is equivalent to digital PCR, and reaches the advanced level of current ctDNA detection technology.

In terms of accuracy, ctDNA has severe fragmentation (average length of only 166 bp), low content (less than 1% of peripheral blood free DNA), and short half-life (about 2 hours). The requirements are extremely high. Among the commercially available ctDNA detection reagents, the accuracy of products based on ARMS-PCR technology is generally about 60-70%, which is far from the clinical demand, thus seriously restricting its clinical application. The human EGFR gene mutation detection kit provided by the invention has a precision of more than 95% in comparison with the biopsy results in the clinical pre-test, and the accuracy of the digital PCR technique is only about 70% in the same period. Therefore, the accuracy is obviously superior to the existing one. Products on the market. (3) low cost

First of all, in terms of detection equipment, fluorescence signal detection is currently used in both ARMS-PCR and digital PCR. The detection equipment needs to be equipped with an expensive fluorescence detection system. Therefore, the market price of the fluorescent quantitative PCR instrument used in ARMS-PCR technology is in the dozens. About 10,000 yuan, and the digital PCR instrument is more than 1 million yuan. As for the high-throughput sequencer used in NGS technology, whether it is optical sequencing represented by illumina or semiconductor sequencing system represented by ion proton, the market price is as high as several million yuan, and the high price severely restricts its Clinical promotion and application. In contrast, the present invention uses an electric field-guided release and measurement technique, the detection process utilizes an electric field, the reaction is fast, and the final result is detected in the form of an electrical signal, so the detection device is not equipped with expensive fluorescence like ARMS-PCR and digital PCR. The detection system, so the cost of the equipment is greatly reduced, only about half of the fluorescence quantitative PCR instrument, less than one tenth of the high-throughput sequencer.

Secondly, in terms of detection reagents, EFIRM technology is based on the principle of nucleic acid hybridization, using a uniquely designed electrochemical technique, the detection probe does not need to be fluorescently labeled like ARMS-PCR, and because of the steps of DNA extraction and purification, The cost of the test reagent is greatly reduced compared to this. Compared with NGS, DNA extraction and purification, library construction, sequencing template preparation, and sequencing are used. Compared with multiple kits, EFIRM's reagent cost is more than 90%, and the competitive advantage is obvious.

(4) significantly reduce the requirements for clinical customer hardware and personnel

Since the use of EFIRM does not require DNA extraction and PCR amplification, there is no problem of easy contamination of PCR. In clinical use, it is not necessary to establish a clinical gene amplification laboratory, and the operator does not need to obtain a clinical gene amplification approval permit. The operation of general technicians makes the threshold of clinical application of liquid biopsy technology greatly reduced, which is conducive to the wide-scale promotion of liquid biopsy technology. The existing ARMS-PCR, digital PCR and NGS technologies can only be carried out in a few large-scale top three hospitals with conditions. The status quo.

DRAWINGS

1 is a schematic plan view showing a structure of a detecting electrode used in the method of the present invention;

2 is a schematic plan view showing another structure of a detecting electrode used in the method of the present invention;

3 is a schematic plan view showing another structure of a detecting electrode used in the method of the present invention;

4 is a schematic plan view showing another structure of a detecting electrode used in the method of the present invention;

Figure 5a is a schematic perspective view of a detection orifice plate used in the method of the present invention;

Figure 5b is a schematic plan view of a detecting orifice plate used in the method of the present invention;

Figure 6 is a partial perspective view of a detecting orifice plate used in the method of the present invention;

Figure 7 is a partial perspective view showing another detecting orifice plate used in the method of the present invention;

Figure 8 is a partial perspective view showing another detecting orifice plate used in the method of the present invention;

Figure 9 is a partial side elevational view of a detection orifice plate employed in the method of the present invention.

detailed description

The embodiments of the present invention will be described in detail below with reference to the accompanying drawings, but the present invention is to be construed as illustrative only. Those who do not specify the specific conditions in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained by commercially available purchase.

The invention is exemplarily described below by way of specific embodiments.

The EFIRM detector used in the present invention is produced by Guangzhou Yihuo Biotech Co., Ltd., and is described in "Felvi Chemical Sensor for Multiplex Biomarkers Detection, Clin Cancer Res. 2009 Jul 1; 15 (13) published by Fang Wei et al. In 4446–4452, the electrochemical detector used therein.

According to the description of the present invention and the prior art mentioned above, a general instrument pair capable of generating a square wave can be employed by those skilled in the art. The square wave (csw E-field) applied to the reaction well can also be realized by using the EFIRMY instrument and related software developed by Yihuo Biotechnology Co., Ltd.

Example 1

The following steps are used to detect different test samples:

1. The capture probe is fixed at the bottom of the detection orifice plate.

1.1 Preparation of a mixture of pyrrole and capture probe (CP):

Take a 1.5ml centrifuge tube, add 885μl of ultrapure water, 100μl of 3M KCl, vortex and mix, centrifuge; add 5μl pyrrole, vortex and mix, centrifuge; add 10μl of 100μM capture probe; vortex mixing After centrifugation, mix and set aside.

1.2 Loading:

On a 96-well assay well plate, add 30 μl of the prepared mixture to each well. When the sample is applied, the tip of the gun is attached to the bottom of the well, but the bottom electrode is not touched. After the addition, tilt or tap the test well to make the liquid. The surface of the electrode in the hole is evenly covered, and then the electric field operation is performed immediately on the EFIRM detector (Guangzhou Yi Living).

1.3 EFIRM electric field treatment:

Select the corresponding column for the experiment on the EFIRM software. The electric field parameters are set to: voltage A: 350 mV, 1 s; voltage B: 950 mV, 1 s; 5 cycles. After the electric field is processed, remove it immediately and clean the detection orifice.

1.4 Detection of orifice plate cleaning:

Select the corresponding experiment column on the washing machine program, select the cleaning program (2bottom, 2top), and choose 2×SSC (0.05% SDS) for the lotion. After the cleaning is completed, proceed to the next sample loading operation.

2. Sample hybridization:

2.1 hybrid buffer pretreatment

The hybrid buffer (using the hybrid buffer 1 in the Summary of the Invention) was treated in a water bath at 90 ° C for 10 min in a water bath and then allowed to cool at room temperature for 20 min.

2.2 Preparation of the sample

The samples were taken out from the -20 ° C refrigerator and thawed in a 4 ° C refrigerator. After complete dissolution, the sample and the hybrid buffer are mixed at a volume ratio of 1:2, vortexed and centrifuged, and the sample can be tested.

2.3 Loading:

On the detection well plate, a blank control buffer, a corresponding concentration of the negative control (WT) and a positive control (MT) were added to the corresponding wells, and the amount of the sample was 30 μl. When the sample is applied, the tip of the gun is attached to the bottom of the hole, but the bottom electrode is not touched. After the addition, the tilting or tapping of the detecting plate allows the liquid to uniformly cover the surface of the electrode in the hole, and then immediately goes to the EFIMR for electric field operation.

2.4 EFIRM electric field treatment:

Select the corresponding column for the experiment on the EFIRM software. The electric field parameters are set to: voltage A: 300 mV, 1 s; voltage B: 500 mV, 1 s; 5 cycles. After the electric field is processed, remove it immediately and clean the detection orifice.

2.5 Detection orifice cleaning:

Select the corresponding experiment column on the washing machine program, select the cleaning program (2bottom, 2top), and choose 2×SSC (0.05% SDS) for the lotion. After the cleaning is completed, the DP loading operation is performed immediately.

3. Detection of hybridization

3.1 DP (detection probe) solution preparation:

The casein/PBS solution containing 2% by weight of casein was taken from a refrigerator at 4 ° C. Take a 1.5 mL centrifuge tube, add 990 μl of casein/PBS, add 10 μl of 100 μM DP, vortex and mix, centrifuge, and set aside.

3.2 Loading:

According to the experimental design, 30 μl of the corresponding DP solution was added to the corresponding hole. When the sample was applied, the tip of the gun was attached to the bottom of the hole, but the bottom electrode was not touched. After the addition, the tilting or tapping of the detecting plate made the liquid evenly cover the surface of the electrode in the hole. Then immediately go to the EFIRM for electric field operation.

3.3 EFIRM electric field treatment:

Select the corresponding column for the experiment on the EFIRM software. The electric field parameters are set to: voltage A: 300 mV, 1 s; voltage B: 500 mV, 1 s; 5 cycles. After the electric field is processed, remove it immediately and clean the detection orifice.

3.4 Detection of orifice cleaning:

Select the corresponding experiment column on the washing machine program, select the cleaning program (2bottom, 2top), and choose 2×SSC (0.05% SDS) for the lotion. After the cleaning is completed, the sample loading operation is performed immediately.

4, Reporter Hybridization

4.1 Poly-HRP solution preparation:

Remove the casein/PBS solution containing 2% by weight of casein from a 4°C refrigerator, take a 1.5mL centrifuge tube, add 999μl casein/PBS (put the casein/PBS solution back to the 4°C refrigerator), and add 1μl of poly -HRP (commercially available from Thermo Fisher, product name Pierce ^TM Streptavidin Poly-HRP, item number 21140, the unit size is 0.5 mL), mixed by vortexing, centrifugation, standby.

4.2 Loading:

According to the experimental design, 30μl of Poly-HRP solution was added to the corresponding hole. When the sample was applied, the tip of the gun was attached to the bottom of the hole, but the bottom electrode was not touched. After the addition, tilt or tap the detection plate to evenly cover the surface of the electrode in the hole. Then, cover the detection plate cover, incubate for 30 minutes at room temperature on the bench, and count down the timer. The incubation plate is cleaned immediately after the incubation time.

4.3 Detection orifice cleaning:

Select the corresponding experiment column on the washing machine program, choose the cleaning program (3bottom, 3top), and choose PBST (0.1% Tween20) for the lotion. After the cleaning is completed, the TMB loading operation is performed immediately.

5, Readout

5.1 Loading:

According to the experimental design, TMB/H2O2 solution (purchased from thermo fisher, product no. 34022, named as

Turbo TMB substrate solution), 60 μl per well, with the tip attached to the bottom of the well while loading, but not touching the bottom electrode. Immediately after the addition, the electric field operation was performed on the EFIRM.

5.2 EFIRM electric field reading:

Select the corresponding column for the experiment on the EFIRM software. The electric field parameter is set to: voltage A: -200mV, 60s, and the current reading is obtained.

In this embodiment, different detection samples include a negative group and a positive group, and the genes determined by the negative group are corresponding wild type sequences, and the genes determined by the positive group are corresponding mutant sequences, and 20 parallel holes are set for each sample for detection.

Correspondingly, the negative group (WT) and the positive group (MT) of each group were tested with corresponding probe pairs to identify the stability of the detection, and the coefficient of variation (CV) of the detected current is shown in Table 2.

In Table 2, the coefficient of variation (cv), also known as the coefficient of dispersion or relative deviation (rsd), is the ratio of the standard deviation to the average, expressed as a percentage, and the formula is: cv=sd/ Mean × 100%.

Table 2 test results

Among them, the probe pairs in Table 2 and Table 1 correspond in turn.

As can be seen from Table 2, the human EGFR gene mutation detecting kit provided by the present invention and the sample for detecting the human EGFR gene mutation using the kit can detect a sequence content as low as 1 pM, and the detection sensitivity is high;

The range of values detected is less than 15%, and some even as low as 3%, and the detection stability is good. In addition, the detection of different concentrations of negative and positive samples, the measured values have a very good linear relationship.

In addition, three independent tests were performed on wild-type sequence samples and mutant sequence samples of serial concentration gradients, and 20 replicate wells were tested for each sample. The numerical results measured by wild samples were judged to be wild, but not met. The numerical results measured by the mutant samples are judged as mutations, and vice versa. The specific results are shown in Table 3.

Table 3 coincidence rate

In addition, 190 cases of salivary samples (70 mutations, 120 wild), 220 plasma samples (85 mutations, 135 wild) and 200 pleural effusions have been confirmed by the method of the present embodiment. The liquid samples (90 cases of mutation and 110 cases of wild) were double-blindly tested on different probe pairs. The test results were compared with the clinical test results. The test results are shown in Table 4.

Table 4 test results

sample	Positive coincidence rate	Negative coincidence rate
plasma	98.8%	100%
saliva	98.6%	100%
Pleural effusion	98.9%	100%

In addition, the test data for different samples is shown in Table 5-7.

Table 5 specific detection of saliva samples

Table 6 specific detection of plasma samples

Table 7 specific detection of pleural effusion samples

Among them, the calculation formulas of the positive coincidence rate and the negative coincidence rate in Table 4 are shown in Table 8.

Table 8 sample statistics

Test results	Positive (mutant)	Negative (wild type)
Positive (mutant)	a	b
Negative (wild type)	c	d

Positive coincidence rate = a / (a + c) × 100%;

Negative coincidence rate = b / (b + d) × 100%.

As can be seen from Table 4-7, both the plasma sample and the saliva sample or the pleural effusion sample have a good clinical test coincidence rate, and the accuracy rate is high, exceeding 95%, meeting the needs of clinical testing applications.

When the above method detects the negative sample and the positive sample, the difference in the detected current values is large, and the different samples can be well distinguished. For example, a negative sample of saliva is detected using a 34 probe pair, and the current value is 41.6 to 65.6 (-nA), while the current value of the positive sample is 88.4 to 141.4 (-nA); correspondingly, the same plasma sample is used. The negative sample obtained current value was 40.1 ~ 63.7 (-nA), while the positive sample current value was 83.6 ~ 139.2.

In addition, the concentration of each component in the kit was changed and the detection was carried out, and the results were basically the same as those described above.

In summary, the human EGFR gene mutation detection kit provided by the invention and the detection of the human EGFR gene mutation using the kit are simple and convenient, the sample is non-invasive, and the required sample is small, and the whole detection process can be completed in half an hour; Good performance, can reach 0.1%, high accuracy, more than 95%; low cost, compared with NGS, cost savings of more than 90%; easy to operate, low requirements for operators.

Example 2

Test the test sample, the specific steps are as follows:

1. The capture probe is fixed at the bottom of the detection orifice plate.

1.1 Preparation of a mixture of thiophene and capture probe (CP):

The capture solution contained the following components: 5% by weight of thiophene, 2 mol/L of NaCl, and 1.5 μmol/L of capture probe.

1.2 Loading:

On a 96-well assay plate, add 20 μl of the prepared mixture of thiophene and CP to each well. When the sample is applied, the tip of the gun is attached to the bottom of the well, but the bottom electrode is not touched. After the addition, the tilt or tapping detection hole is applied. The plate evenly covers the surface of the electrode in the hole and immediately goes to the EFIRM detector for electric field operation.

1.3 EFIRM electric field treatment:

The corresponding column for the experiment was selected on the EFIRM software. The electric field parameters were set to: voltage A: 500 mV, 1 s; voltage B: 1500 mV, 1 s; 10 cycles were performed. After the electric field is processed, remove it immediately and clean the detection orifice.

1.4 Detection of orifice plate cleaning:

Select the corresponding experiment column on the washer program, select the cleaning program (2bottom, 2top), and choose 2XSSC (0.06% SDS) for the lotion. After the cleaning is completed, proceed to the next sample loading operation.

2. Sample hybridization:

2.1 hybrid buffer pretreatment

The hybrid buffer (using the hybrid buffer 3 in the Summary of the Invention) was treated in a water bath at 95 ° C for 5 min in a water bath and then allowed to cool at room temperature.

2.2 Preparation of the sample

The samples were taken out from the -20 ° C refrigerator and thawed in a 4 ° C refrigerator. After complete dissolution, the sample is mixed with the hybrid buffer by a volume ratio of 1:2.5, vortex After centrifugation and centrifugation, the sample can be tested.

2.3 Loading:

On the detection well plate, a blank control buffer, a corresponding concentration of the negative control (WT) and a positive control (MT) were added to the corresponding wells, and the sample volume was 80 μl. When the sample is applied, the tip of the gun is attached to the bottom of the hole, but the bottom electrode is not touched. After the addition, the tilting or tapping of the detecting plate allows the liquid to uniformly cover the surface of the electrode in the hole, and then immediately goes to the EFIMR for electric field operation.

2.4 EFIRM electric field treatment:

The corresponding column for the experiment was selected on the EFIRM software. The electric field parameters were set to: voltage A: 500 mV, 1 s; voltage B: 800 mV, 1 s; 10 cycles were performed. After the electric field is processed, remove it immediately and clean the detection orifice.

2.5 Detection orifice cleaning:

Select the corresponding experiment column on the washer program, select the cleaning program (2bottom, 2top), and choose 2XSSC (0.06% SDS) for the lotion. After the cleaning is completed, the DP loading operation is performed immediately.

3. Detection of hybridization

3.1 DP (detection probe) solution preparation:

The detection solution was PBS as a solvent, wherein the weight percentage of casein was 5%, the concentration of the detection probe was 1.5 μmol/L, vortexed and mixed, centrifuged, and set aside.

3.2 Loading:

According to the experimental design, 20 μl of the corresponding DP solution was added to the corresponding hole. When the sample was applied, the tip of the gun was attached to the bottom of the hole, but the bottom electrode was not touched. After the addition, the tilting or tapping of the detecting plate made the liquid evenly cover the surface of the electrode in the hole. Then immediately go to the EFIRM for electric field operation.

3.3 EFIRM electric field treatment:

The corresponding column for the experiment was selected on the EFIRM software. The electric field parameters were set to: voltage A: 500 mV, 1 s; voltage B: 800 mV, 1 s; 8 cycles were performed. After the electric field is processed, remove it immediately and clean the detection orifice.

3.4 Detection of orifice cleaning:

Select the corresponding experiment column on the washer program, select the cleaning program (2bottom, 2top), and choose 2XSSC (0.06% SDS) for the lotion. After the cleaning is completed, the sample loading operation is performed immediately.

4, Reporter Hybridization

4.1 Poly-HRP solution preparation:

With PBS as a solvent, wherein the weight percentage of casein is 5%, the volume percentage of poly-HRP 0.2% (available from Thermo Fisher, product name Pierce ^TM Streptavidin Poly-HRP, item number 21140, the unit size of 0.5 mL) Vortex and mix well, centrifuge, and set aside.

4.2 Loading:

According to the experimental design, 20 μl of Poly-HRP solution was added to the corresponding hole. When the sample was applied, the tip of the gun was attached to the bottom of the hole, but the bottom electrode was not touched. After the addition, the tilting or tapping of the detecting plate allowed the liquid to uniformly cover the surface of the electrode in the hole. Then, cover the detection plate cover, incubate for 40 minutes at room temperature on the bench, and count down the timer. The incubation plate is cleaned immediately after the incubation time.

4.3 Detection orifice cleaning:

Select the corresponding experiment column on the washing machine program, select the cleaning program (3bottom, 3top), and choose PBST (0.15% Tween20) for the lotion. After the cleaning is completed, the TMB loading operation is performed immediately.

5, Readout

5.1 Loading:

According to the experimental design, TMB/H2O2 solution (purchased from thermo fisher, product no. 34022, named as

Turbo TMB substrate solution), 80 μl was added to each well, and the tip of the gun was attached to the bottom of the well while the sample was applied, but did not touch the bottom electrode. Immediately after the addition, the electric field operation was performed on the EFIRM.

5.2 EFIRM electric field reading:

Select the corresponding column for the experiment on the EFIRM software. The electric field parameter is set to: voltage A: -300mV, 100s, and the current reading is obtained.

Example 3

Test the test sample, the specific steps are as follows:

1. The capture probe is fixed at the bottom of the detection orifice plate.

1.1 Preparation of a mixture of aniline and capture probe (CP):

The capture solution contained the following components: the weight percentage of aniline was 0.1%, the concentration of NaCl was 0.01 mol/L, and the concentration of the capture probe was 0.5 μmol/L.

1.2 Loading:

On a 96-well assay plate, add 80 μl of the prepared aniline and CP mixture to each well. When the sample is applied, the tip of the gun is attached to the bottom of the well, but the bottom electrode is not touched. After the addition, the tilt or tapping detection hole is applied. The plate evenly covers the surface of the electrode in the hole and immediately goes to the EFIRM detector for electric field operation.

1.3 EFIRM electric field treatment:

The corresponding column for the experiment was selected on the EFIRM software. The electric field parameters were set to: voltage A: 200 mV, 5 s; voltage B: 800 mV, 5 s; 3 cycles were performed. After the electric field is processed, remove it immediately and clean the detection orifice.

1.4 Detection of orifice plate cleaning:

Select the corresponding experiment column on the washer program, select the cleaning program (2bottom, 2top), and choose 2XSSC (0.04% SDS) for the lotion. After the cleaning is completed, proceed to the next sample loading operation.

2. Sample hybridization:

2.1 hybrid buffer pretreatment

The hybrid buffer (using the hybrid buffer 6 in the Summary of the Invention) was treated in a water bath at 85 ° C for 15 min in a water bath and then left to cool at room temperature.

2.2 Preparation of the sample

The samples were taken out from the -20 ° C refrigerator and thawed in a 4 ° C refrigerator. After complete dissolution, the sample and the hybrid buffer are mixed at a volume ratio of 1:1.5, vortexed and centrifuged, and the sample can be tested.

2.3 Loading:

On the detection well plate, a blank control buffer, a corresponding concentration of the negative control (WT) and a positive control (MT) were added to the corresponding wells, and the amount of the sample was 20 μl. When the sample is applied, the tip of the gun is attached to the bottom of the hole, but the bottom electrode is not touched. After the addition, the tilting or tapping of the detecting plate allows the liquid to uniformly cover the surface of the electrode in the hole, and then immediately goes to the EFIMR for electric field operation.

2.4 EFIRM electric field treatment:

The corresponding column for the experiment was selected on the EFIRM software. The electric field parameters were set to: voltage A: 200 mV, 5 s; voltage B: 300 mV, 5 s; 3 cycles were performed. After the electric field is processed, remove it immediately and clean the detection orifice.

2.5 Detection orifice cleaning:

Select the corresponding experiment column on the washer program, select the cleaning program (2bottom, 2top), and choose 2XSSC (0.04% SDS) for the lotion. After the cleaning is completed, the DP loading operation is performed immediately.

3. Detection of hybridization

3.1 DP (detection probe) solution preparation:

The detection solution was PBS as a solvent, wherein the weight percentage of casein was 0.1%, the concentration of the detection probe was 0.5 μmol/L, vortexed and mixed, centrifuged, and set aside.

3.2 Loading:

According to the experimental design, 30 μl of the corresponding DP solution was added to the corresponding hole. When the sample was applied, the tip of the gun was attached to the bottom of the hole, but the bottom electrode was not touched. After the addition, the tilting or tapping of the detecting plate made the liquid evenly cover the surface of the electrode in the hole. Then immediately go to the EFIRM for electric field operation.

3.3 EFIRM electric field treatment:

The corresponding column for the experiment was selected on the EFIRM software. The electric field parameters were set to: voltage A: 200 mV, 5 s; voltage B: 300 mV, 5 s; 3 cycles were performed. After the electric field is processed, remove it immediately and clean the detection orifice.

3.4 Detection of orifice cleaning:

Select the corresponding experiment column on the washer program, select the cleaning program (2bottom, 2top), and choose 2XSSC (0.04% SDS) for the lotion. After the cleaning is completed, the sample loading operation is performed immediately.

4, Reporter Hybridization

4.1 Poly-HRP solution preparation:

With PBS as a solvent, wherein the weight percentage of casein 0.1%, the volume percentage of poly-HRP in 0.05% (available from Thermo Fisher, product name Pierce ^TM Streptavidin Poly-HRP, item number 21140, the unit size of 0.5 mL) Vortex and mix well, centrifuge, and set aside.

4.2 Loading:

According to the experimental design, add 20-80μl of Poly-HRP solution to the corresponding hole. When the sample is applied, the tip of the gun is close to the bottom of the hole, but it does not touch the bottom electrode. After the addition, tilt or tap the surface of the electrode to detect the hole in the hole. Cover evenly, then cover the detection plate cover, incubate for 20-40min at room temperature on the bench, and count down the timer. The incubation plate is cleaned immediately after the incubation time.

4.3 Detection orifice cleaning:

Select the corresponding experiment column on the washer program, select the cleaning program (3bottom, 3top), and choose PBST (0.05% Tween20) for the lotion. After the cleaning is completed, the TMB loading operation is performed immediately.

5, Readout

5.1 Loading:

According to the experimental design, TMB/H ₂ O ₂ solution was added to the corresponding well (purchased from thermo fisher, product No. 34022, the name is

Turbo TMB substrate solution), add 30 μl to each well, and attach the tip to the bottom of the well while loading, but not the bottom electrode. Immediately after the addition, the electric field operation was performed on the EFIRM.

5.2 EFIRM electric field reading:

Select the corresponding column for the experiment on the EFIRM software. The electric field parameter is set to: voltage A: -100mV, 40s, and the current reading is obtained.

Similarly, the methods provided in Examples 2 and 3 of the present invention were tested in the same manner as in Example 1, and the results obtained were basically in agreement with those in Example 1.

It can be seen that the human EGFR gene mutation detection kit provided by the invention and the detection of the human EGFR gene mutation using the kit are simple and convenient, the sample is non-invasive, and the required sample is small, and the whole detection process can be completed in half an hour; the sensitivity is good. , can reach 0.1%, high accuracy, more than 95%; low cost, compared with NGS, cost savings of more than 90%; easy to operate, low requirements for operators.

The detection orifice plate used in the method of the invention has the common feature that the inner bottom of the reaction well is provided with an electrode for applying an electric field to the solution in the reaction well after the EFIRM detector is turned on. Existing commercial products can be used.

In some preferred embodiments of the present invention, a detecting orifice structure is adopted, wherein the detecting electrode structure is arranged as shown in FIG. 1: comprising: a reaction well bottom plate 101, the reaction well bottom plate 101 includes at least one detecting region 102; and the working electrode 103 The working electrode 103 is disposed on the reaction well substrate 101 and configured to apply a voltage to form an electric field; and the opposite electrode 104 is disposed on the reaction aperture substrate 101 and configured to acquire a detection signal and output the detection signal. For example, the working electrode 103 and the opposite electrode 104 are both disposed on the same surface of the reaction cell bottom plate, and therefore, the working electrode 103 and the opposite electrode 104 may be in the same plane. As shown in FIG. 1, the working electrode 103 includes at least one first linear portion 1031 having a uniform width; the opposite electrode 104 includes at least one second linear portion 1041 of uniform width; the first linear portion 1031 and the second linear portion 1041 They are disposed within the detection zone 102 and are alternately spaced apart from one another. It should be noted that the above-mentioned working electrode 103 and the opposite electrode 104 have the same structure. Therefore, the electrode 104 located on the right side of FIG. 1 can be configured as a working electrode to apply a voltage to form an electric field; the electrode 103 located on the left side of FIG. 1 can be The counter electrode is configured to acquire a detection signal and output the detection signal, and the disclosure is not limited herein.

In the structure of the detecting electrode provided in this embodiment, the working electrode 103 can apply a voltage to generate an electric field to move and concentrate the target substance. For example, the working electrode 103 can apply a square wave alternating voltage to first include the target substance in the liquid to be detected. The charged substance moves to the working electrode 103 to be enriched, so that the target substance can be combined with the probe on the working electrode 103, and then the polarity of the voltage is changed, so that other substances in the charged substance that are not combined with the probe are away from the working electrode 103 ( The force of the electric field on the target substance is set to be smaller than the binding force of the target substance and the probe; then, the opposite electrode 104 can acquire a detection signal about the target substance and output the detection signal, for example, a target substance bound to the probe. The current reacts with a specific reagent to generate a current, so the opposite electrode 104 can acquire a detection signal about the target substance by detecting the current and output the detection signal; and then, by analyzing the output detection signal, the target substance can be obtained. Information (such as the concentration of the target substance) so that it can be detected quickly and accurately. Since the first linear portion 1031 and the second linear portion 1041 are linear structures having a uniform width, and within the detection region 102, the first linear portion 1031 and the second linear portion 1041 are alternately equidistantly spaced within the detection region 102. Therefore, the first linear portion 1031 of the working electrode 103 can generate a uniform electric field in the detection region 102, and the second linear portion 1041 of the opposite electrode 104 can detect a minute current in the detection region 102, thereby improving The accuracy of the detection. In addition, since the first linear portion 1031 of the working electrode 103 and the second linear portion 1041 are alternately spaced, the density and uniformity of the probe formed on the working electrode 103 can be controlled, and the probe is not excessively dense. Thereby providing space for the binding of the target substance and the probe, and improving the efficiency of binding of the target substance and the probe, thereby improving the reaction speed of the liquid biopsy and further improving the detection accuracy.

As shown in FIG. 1, the detecting electrode structure in the detecting orifice plate has a circular shape, and the working electrode 103 may include an arc-shaped first main body portion 1030 and a plurality of extending from the first main body portion 1030. The first linear portion 1031 that is parallel to each other. The counter electrode 104 includes an arc-shaped second body portion 1040 and a plurality of second linear portions 1041 that are parallel to each other and extend from the second body portion 1040. The first body portion 1030 is disposed opposite to the second body portion 1040, and the plurality of first linear portions 1031 and the plurality of second linear portions 1041 are disposed in the detection region 102 and are alternately spaced and equidistantly disposed. That is, the working electrode 103 and the opposite electrode 104 have a comb-like structure, and the working electrode 103 and the opposite electrode 104 cross each other to form an interdigitated structure. It should be noted that the range of the detection area may include a plurality of first linear portions and a plurality of second linear portions, and may further include a first main body portion and a second main body portion, which are not limited herein. The working electrode may not be provided with a fixing portion that is in direct contact with the probe, so the first linear portion may be formed into a line having a uniform width, thereby providing a more uniform electric field, so that the arrangement of the probe is more regular, thereby improving the efficiency and accuracy of detection. .

2 is a schematic plan view showing another structure of the detecting electrode. The detecting area 102 has a circular shape, the working electrode 103 includes a first linear portion 1031 which is spirally arranged, and the opposite electrode 104 includes a second line which is spirally arranged. The portion 1041, the first linear portion 1031 and the second linear portion 1041 are disposed in the detection region 102 and are alternately spaced and equidistantly disposed. In the detecting electrode structure provided by the example of the embodiment, the width of the first linear portion is the same as the width of the second linear portion, so that the accuracy of the detection can be improved; in addition, the width of the first linear portion and the second linear portion The range can be 3-20 mils (thousandths of an inch).

For example, in the detecting electrode structure provided in the example of the embodiment, the pitch of the first linear portion and the second linear portion may range from 3 to 20 mils (thousandths of an inch).

For example, in the detecting electrode structure provided in the example of the embodiment, the width of the first linear portion and the width of the second linear portion are equal to the spacing between the first linear portion and the second linear portion. Thereby, the uniformity of the electric field and the uniformity of detection can be further improved, so that the accuracy of detection can be improved.

For example, FIG. 3 is a schematic plan view of another detecting electrode structure, and FIG. 4 is a schematic plan view of another detecting electrode structure. As shown in FIG. 3 or FIG. 4, the detecting electrode structure provided in an example of the embodiment further includes setting. At the reference electrode at the edge of the detection zone, since the reference electrode is disposed at the edge of the detection zone, the outer side of the first linear portion and the second linear portion, the reference electrode can provide a contrast in the process of acquiring the detection signal about the target substance. The polarity error of the working electrode is eliminated, thereby further improving the accuracy of the detection.

For example, in the detecting electrode structure of the detecting orifice plate used in another embodiment, the material of the working electrode and the opposing electrode includes gold. Since the chemical nature of the gold element is stable and does not react with the liquid to be detected and has a lower impedance, the accuracy of the detection can be further improved. Of course, including but not limited to, other conductive materials such as platinum or indium tin oxide may also be used.

5a and 5b illustrate a detection orifice plate used in the method of the present invention, the detection orifice plate comprising: a cartridge body 200 and a detection electrode structure 100, the cartridge body 200 comprising a plurality of reaction wells 211, the size of the reaction wells Reference may be made to the design of a conventional 96-well plate. Of course, the present disclosure includes but is not limited thereto, and the size of the reaction well may be designed according to the concentration and kind of the liquid to be detected. As shown in FIGS. 6 and 7, the detecting electrode structure 100 is disposed at the bottom of the casing 200, the detecting electrode structure 100 may be the detecting electrode structure of any of the above-described first embodiment, and the detecting region 102 is disposed at the bottom of the reaction hole 211. The bottom of the reaction well 211 is sealed. Further, the reaction cell bottom plate 101 of the detecting electrode structure 100 and the casing 200 can be made of the same material. Thereby, the liquid to be detected can be contained in the accommodating space composed of the detection zone 102 and the reaction well 211, thereby detecting the liquid to be detected. For example, the working electrode 103 may apply a square wave alternating voltage to form a vertical electric field perpendicular to the bottom surface of the through hole 211, first causing the charged substance including the target substance in the liquid to be detected from the respective positions of the reaction hole 211 to the reaction hole 211. The bottom moves and moves to the working electrode 103 to enrich, so that the target substance can be combined with the probe on the working electrode 103 (a substance that can bind to the target substance, such as a DNA polymer molecule), and then the polarity of the voltage is converted to make it perpendicular to the pass. The direction of the vertical electric field on the bottom surface of the hole 211 is reversed, so that other substances not charged with the probe in the charged substance in the liquid to be detected move from the bottom of the reaction hole 211 to the upper portion of the reaction hole 211, thereby causing other substances in the charged substance. The substance not bound to the probe is away from the working electrode 103 (the force of the electric field on the target substance is set to be smaller than the binding force of the target substance and the probe); then, the opposite electrode 104 can acquire the detection signal about the target substance and detect The signal output, for example, the target substance bound to the probe reacts with a specific reagent to generate a current, so the opposite electrode 104 can pass the inspection. The current is measured to obtain a detection signal about the target substance and the detection signal is output; then, the information about the target substance (for example, the concentration of the target substance) can be obtained by analyzing the output detection signal.

For example, in the detecting orifice plate provided by the present invention, as shown in FIG. 5b, a plurality of reaction wells 211 are arranged in a matrix in the cartridge body 200. Figure 6 Or as shown in FIG. 7, the plurality of reaction holes 211 are cylindrical through holes. It should be noted that the shape of the reaction well includes, but is not limited to, the shape of the plurality of reaction holes 211 may also be a square cylinder, a triangular cylinder or other cylinders.

For example, in the detection orifice plate used in the present method, the number of the plurality of reaction holes 211 is a multiple of four, as shown in FIG. 8, four adjacent reaction holes 211 correspond to the four working electrodes 103 in the detection electrode structure. Electrically connected. For example, the reaction hole bottom plate 101 is formed with a wire 111 and a wire 112. The wire 111 electrically connects the four working electrodes 103. The wires 112 are electrically connected to the four opposite electrodes 104 to extract the electrical signals of the opposite electrode 104. . Thus, the four adjacent through holes can be used as a detection group. In the detection group, since the four working electrodes are electrically connected, the voltages applied to the four working electrodes are uniform, and four adjacent through holes are The control experiment can be performed better, so the detection accuracy can be further improved. Of course, any number of working electrodes in the detection electrode structure corresponding to any number of through holes may be electrically connected to provide a uniform voltage.

For example, as shown in FIG. 9, the detection aperture board used in the present invention further includes: a circuit board 110 electrically connected to the detection pole structure. An amplification circuit may be disposed on the circuit board 110 to amplify the electrical signal outputted by the opposite electrode or the reference electrode to improve detection accuracy; a voltage stabilization circuit may also be disposed on the circuit board 110 to provide a stable voltage to the working electrode to improve detection accuracy. Of course, the present disclosure includes but is not limited thereto, and an overcurrent, overvoltage protection circuit, or the like may be disposed on the circuit board 110.

As shown in FIG. 9, the circuit board 110 can be disposed under the reaction hole bottom plate 101, so that the space can be utilized more reasonably. Of course, the circuit board 110 can also be disposed at other positions, and the disclosure is not limited herein.

While the invention has been illustrated and described with reference to the embodiments of the present invention, it will be understood that many modifications and changes can be made without departing from the spirit and scope of the invention. Accordingly, it is intended to embrace in the appended claims

Claims (20)

1. a probe combination for detecting a mutation of a human EGFR gene, characterized in that the probe comprises at least one of a pair of 1-35 probe pairs, each of which comprises a capture probe and a detection probe;

   The nucleotide sequence of the 1-35 probe pair is shown in SEQ ID NO: 1-70;

   Among them, the 1-3 probe pair is used to detect the exon 18 point mutation of EGFR, the 4-28 probe pair is used to detect the exon 19 deletion mutation of EGFR, and the 29-33 probe pair is used for detecting EGFR. The mutant 20 mutation, the 34-35 probe pair was used to detect the exon 21 point mutation of EGFR.
2. The probe assembly according to claim 1, wherein the detection probe is provided with a label, and the label is preferably any of digoxin, biotin, and fluorescein isothiocyanate. One.
3. A detection plate for detecting mutation of human EGFR gene based on EFIRM technology, characterized in that: the bottom of the reaction hole is provided with an electrode for applying an electric field to the solution in the reaction hole after the EFIRM detector is turned on, and the reaction hole is distributed And a capture probe in the probe combination of claim 1 is immobilized, and each of the reaction wells is immobilized with one of the capture probes.
4. The detection plate for detecting mutation of a human EGFR gene according to claim 3, wherein the capture probe is mixed with the immobilizer and adhered to the reaction well of the detection plate by an electric field;

   The fixture comprises a conductive polymer material and a salt ion compound;

   The conductive polymer material is selected from at least one of aniline, thiophene and pyrrole conductive molecular monomers;

   The salt ionic compound is at least one selected from the group consisting of a chloride salt, a nitrate salt, and a sulfate salt.
5. The method for preparing a detection plate for detecting a mutation of a human EGFR gene according to claim 3 or 4, wherein the capture probe is mixed with the immobilization as a capture solution,

   Adding the capture solution to the reaction well of the blank detection well plate, and the bottom of the reaction well is provided with an electrode for turning on the EFIRM detector to apply an electric field to the solution in the reaction well for polymerization;

   After the EFIRM detector is turned on, an electric field is applied to the solution in the reaction well to carry out polymerization.
6. The preparation method according to claim 5, wherein the composition of the capturing solution is as follows: the volume percentage of the conductive polymer material is 0.1% to 5%, and the concentration of the salt ion compound is 0.01 mol/L to 2 mol/L. The concentration of the capture probe is 0.5-1.5 μmol/L. The conductive polymer material is selected from at least one of aniline, thiophene and pyrrole conductive molecular monomers;

   The salt ionic compound is selected from at least one of a chloride salt, a nitrate salt, and a sulfate salt;

   The parameters of the electric field are as follows: voltage A: 350 mV, 1 s; voltage B: 950 mV, 1 s; 5 cycles are performed.
7. A human EGFR gene mutation detecting kit based on EFIRM technology, characterized in that it comprises at least one or more of the probe set of claim 2, an enzyme, and a substrate corresponding to the enzyme;

   The enzyme is labeled with a substance that binds to a label provided on the detection probe.
8. The human EGFR gene mutation detecting kit according to claim 7, wherein the detection probe is provided with a combination of a label and a combination of the label in any one of the following groups:

   The label set on the detection probe is digoxin or fluorescein isothiocyanate, and the enzyme-labeled conjugate is correspondingly a digoxin antibody or a fluorescein isothiocyanate antibody;

   The label set on the detection probe is biotin, and the enzyme-labeled conjugate is avidin.
9. The human EGFR gene mutation detecting kit according to claim 7 or 8, wherein the enzyme is a horseradish peroxidase polymer or an alkaline phosphatase;

   When the enzyme is a horseradish peroxidase polymer, the substrate is selected from the group consisting of a mixture of TMB and H ₂ O ₂ , o-phenylenediamine, ABTS, 3-(4-hydroxy);

   When the enzyme is alkaline phosphatase, the substrate is selected from the group consisting of p-nitrophenyl phosphate, a combination of BCIP and NBT, disodium 4-nitrophenyl phosphate, naphthol AS-BI phosphate, naphthol-AS a group consisting of -MX-phosphate.
10. The human EGFR gene mutation detecting kit according to any one of claims 7 to 9, further comprising a fixing substance for immobilizing the capturing probe, a hybridization buffer, a blocking protein, a PBS, a washing solution, and a detecting hole. board;

    The blocking protein is preferably any one of casein, fetal bovine serum, and calf serum.
11. The human EGFR gene mutation detecting kit according to claim 10, wherein the immobilizer comprises a conductive polymer material and a salt ion compound;

    The conductive polymer material is any one of aniline, thiophene and pyrrole conductive molecular monomers;

    The salt ionic compound is any one of a chloride salt, a nitrate salt and a sulfate salt, and the chloride salt is sodium chloride, potassium chloride, magnesium chloride, and chlorination. One of ammonium, the nitrate is one of sodium nitrate, potassium nitrate, magnesium nitrate, and ammonium nitrate, and the sulfate is one of sodium sulfate, potassium sulfate, magnesium sulfate, and ammonium sulfate.
12. The human EGFR gene mutation detecting kit according to claim 11, wherein the capturing probe and the immobilizer are present in the form of a trapping solution containing the following components: a volume of the conductive polymer material The percentage is from 0.1% to 5%, the concentration of the salt ionic compound is from 0.01 mol/L to 2 mol/L, and the concentration of the capture probe is from 0.5 to 1.5 μmol/L.
13. The human EGFR gene mutation detecting kit according to any one of claims 10 to 12, further comprising the detecting orifice plate according to claim 3 or 4, that is, the capturing probe and the fixing object are attached It exists in the form of the reaction well of the detection orifice.
14. The human EGFR gene mutation detecting kit according to any one of claims 7 to 13, characterized in that the detecting probe is present in the form of a detection solution;

    The detection solution is PBS as a solvent, wherein the weight percentage of the blocking protein is 0.1% to 5%, and the concentration of the detection probe is 0.5 to 1.5 μmol/L.
15. The human EGFR gene mutation detecting kit according to any one of claims 7 to 13, characterized in that the enzyme is in the form of an enzyme solution, wherein the enzyme solution is PBS as a solvent, wherein the weight percentage of the blocked protein It is 0.1% to 5%, and the volume percentage of the enzyme is 0.05% to 0.2%.
16. The human EGFR gene mutation detecting kit according to claim 10, wherein the cleaning solution comprises a 0.1-2×SSC mixture containing a mass concentration of 0.04% to 0.06% SDS and a volume percentage of 0.05%- 0.15% Tween 20 in PBS.
17. A method for detecting a mutation in a human EGFR gene based on EFIRM, comprising the steps of:

    (1) using the detection orifice plate of claim 3 or 4, or

    The capture probe comprising the probe set according to claim 1 or 2 is added to a blank detection well plate, and the bottom of the reaction well is provided with an electrode for applying the solution in the reaction well after the EFIRM detector is turned on. The electric field is subjected to a polymerization reaction; after the EFIRM detector is turned on, a first electric field is applied to the solution in the reaction well to carry out a polymerization reaction; after the electric field treatment is completed, the detection orifice plate is cleaned;

    The parameters of the first electric field processing are: voltage 200-500 mV, 1-5 s; voltage 800-1500 mV, 1-5 s; 3-10 cycles;

    (2) adding a mixed solution of the sample to be tested and the hybrid buffer, applying a second electric field to the reaction well: voltage 200-500 mV, 1-5 s; voltage 300-800 mV, 1-5 s; 3-10 cycles; cleaning detection orifice ;

    (3) adding a detection solution, applying a third electric field: voltage 200-500 mV, 1-5 s; voltage 300-800 mV, 1-5 s; 3-8 cycles; cleaning the detection orifice;

    The detection solution contains the detection probe in the probe set according to claim 1 or 2, and the concentration is 0.5-1.5 μmol/L;

    (4) adding the enzyme solution, washing after incubation;

    (5) adding a substrate and reading under an electric field voltage of -100 to -300 mV to obtain a current value;

    The sample to be tested is a body fluid, and the body fluid includes plasma, saliva, and pleural effusion.
18. The method according to claim 17, wherein said enzyme solution is PBS as a solvent, wherein the weight percentage of the blocking protein is from 0.1% to 5%, and the volume percentage of the enzyme is from 0.05% to 0.2%.
19. The method according to claim 17 or 18, wherein the cleaning liquid comprises a 0.1-2 x SSC mixture containing a mass concentration of 0.04% to 0.06% SDS and a volume percentage of 0.05% to 0.15% Tween20. PBS solution.
20. The method according to any one of claims 17 to 19, wherein the detection solution is PBS as a solvent, wherein the weight percentage of the blocking protein is 0.1% to 5%, and the concentration of the detection probe is 0.5- 1.5 μmol/L.

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