WO2009129693A1 - Probes, liquidchip-based products, and methods for detection of egfr gene mutation - Google Patents

Abstract

Probes, liquidchip-based products, and methods for detection of EGFR gene mutation are disclosed. The liquid chip for detection of EGFR gene mutation mainly includes: microspheres coated with probes; primers for amplifying target sequence in exon 19 and/or exon 21.The liquid chip and methods can detect mutations with relatively higher frequency in EGFR gene, and detect mutations in exon 19 and 21 simultaneously or separately. The assay has high specificity and sensitivity with uniform reaction condition. Detection has over 90 percent accuracy and takes less time. The method can be applied not only to detection of EGFR gene mutations in tumor tissue samples but also to those in humour samples from cancer patients. Thus it can monitor dynamicly.

Description

 Detection probe for EGFR gene mutation site, liquid phase chip and detection method thereof

 The invention belongs to the field of molecular biology, and relates to medicine and biotechnology, and specifically relates to a detection probe, a liquid phase chip and a detection method thereof for a mutation site of an EGFR gene.

technical background

 Epidermal growth factor receptor (EGFR) is a multifunctional glycoprotein widely distributed on the cell membrane of human tissues. It has tyrosine kinase activity and is one of the four members of the ErbB family. HER1 or ErbBl. Upon activation of the ligand, EGFR initiates intracellular signaling, undergoes a cascade of adaptor proteins and enzymes in the cytoplasm, regulates transcription of transcription factor-activated genes, and directs cell migration, adhesion, proliferation, differentiation, and apoptosis. Studies have shown that the high expression or abnormal expression of EGFR in many solid tumors provides a theoretical basis and experimental basis for EGFR-targeted tumor therapy and signal transduction intervention for EGFR signaling pathway. Therefore, molecular targeted therapy with epidermal growth factor receptor (EGFR) as a therapeutic target has become the focus of attention in the oncology community at home and abroad. Among them, EGFR receptor tyrosine kinase inhibitors gefitinib (Genfitinib, Irressa) and erlotinib (Erlotinib, Tarceva, Tarceva) have been approved by the US Food and Drug Administration for the treatment of advanced non-small cells. Lung cancer (NSCLC). In February 2005, China also approved gefitinib to enter the clinic. These drugs are used in the treatment of other tumors (such as breast cancer, colon cancer, and gastric cancer) and are now in clinical trials.

 Gefitinib, erlotinib is an oral small molecule EGFR antagonist that competes with ATP for binding to the ATP-binding pocket of the EGFR tyrosine kinase domain to inhibit EGFR activity. It has been used in advanced and unsuitable for traditional chemotherapy. The clinical treatment of patients with non-small cell lung cancer is currently the most effective treatment for some patients with NSCLC. However, the patient's responsiveness to this molecularly targeted drug is related to the individual's genetic background. In June 2004, researchers at Harvard Medical School in the United States, Lynch et al. and Paez, etc., first reported that mutations in the EGFR tyrosine kinase coding region in lung cancer cells are a necessary prerequisite for targeted therapeutics. The results showed that the efficiency of gefitinib was more than 80% for mutant tumors with EGFR tyrosine kinase gene coding region, and the above drugs were basically ineffective for wild-type tumors without mutation. After the publication of this latest research, it has been highly valued by scholars from all over the world, and has been confirmed by research institutes of the United States, Japan, South Korea and China (including Taiwan) scholars in less than one year (Pao W et al., 2004; Han SW Et al., 2005; Mitsudomi T et al., 2005; Huang SF et al., 2004; Mu XL et al., 2005). Therefore, before the treatment with gefitinib, erlotinib or the like, the detection of the presence of relevant EGFR functional mutations can accurately predict the effectiveness of molecularly targeted drug therapy, facilitate clinical accurate drug use, and significantly improve drug efficacy. To maximize the benefit of patients, while avoiding the increase in medical expenses for patients and the waste of social medical resources caused by irrational use of drugs, reducing the aging loss of unnecessary treatment and economic losses.

Currently, the detection standard for EGFR (tyrosine kinase domain) gene mutations is the direct sequencing of tissue DNA samples. The method was first reported by Lynch et al. and Paez et al. The advantage is that the range and type of EGFR gene mutation can be accurately understood, and the relationship between gene mutation and the clinical efficacy of gefitinib has been affirmed. However, gene sequencing as a clinical routine diagnosis method also has many obstacles to be excluded. First, the detection of trace tissue specimens is difficult. Tissue DNA is amplified by PCR and then gene ten Sequencing, DNA requirements are at least 100 ng or more. At present, the specimens for detecting EGFR gene mutations are mainly taken from postoperative tissue samples. However, 70%-80% of cancer patients have difficulty in radical resection at the time of diagnosis, and tissue specimens cannot be obtained for EGFR gene mutation determination. The only way to get tissue in advanced cancer patients is through a biopsy. However, the sample of tumor biopsy tissue is small. Usually only contains tens of thousands of cells. Therefore, the detection of EGFR genes in micro-tissue specimens is quite difficult. The EGFR gene detection of such specimens has not been successfully reported in China. Secondly, tumor biopsy surgery is only suitable for some patients, and specimen collection is more difficult. Advanced tumors are mostly in a high-risk state of rupture and hemorrhage, and there is a certain medical risk in needle biopsy as a kind of injury surgery. And a considerable number of patients are not suitable for this operation because of the special anatomical location of the lesion (such as brain metastasis, lesions adjacent to the heart and large blood vessels, etc.). Lead to the failure to provide tumor tissue for genetic diagnosis. Finally, this method requires high technical requirements and results in time-consuming and laborious interpretation. Although gene sequencing is the most intuitive and accurate detection method, the scope and type of mutation can be clearly diagnosed. However, the detection period is long. It takes 15-21 hours for the report to be sent from the sample to be tested to the successful completion of the test. If the test is performed under normal working hours, it usually takes at least 3 working days to issue a test result report. Therefore, it is difficult to meet the requirements for guiding the timeliness of clinical use. At the same time, since more than 95% of the mutations belong to heterozygous mutations (that is, both wild-type and mutant amplification products exist), it is difficult to accurately diagnose the mutation types, especially the gene mutation spectrum of exon 19 is complicated. Specimens need to be cloned to determine the type of mutation. Therefore, the method is only suitable for a small number of well-equipped laboratories. The existing research in China is based on key universities and research institutions (such as the Chinese Academy of Sciences and the Union Medical University). Hospitals at all levels rarely carry out this diagnosis independently.

 Therefore, the detection of EGFR gene mutations should become a clinical routine diagnostic technique. There are two key issues that need to be solved urgently: 1 It is necessary to find a suitable surrogate source for tumor patients, so that patients with advanced cancer do not have to obtain materials from tumor tissues. Can also enter the EGFR gene diagnosis. 2 It is necessary to develop new detection technologies with good specificity, high sensitivity and easy implementation, so that hospitals at all levels can carry out this genetic diagnosis project.

 Therefore, the establishment of a EGFR gene mutation diagnostic method with convenient sampling, small patient pain, good specificity, high sensitivity, accuracy, rapidity, simplicity and no need for tissue specimens is an important problem to be solved in the treatment of EGFR TKIs in NSCLC patients.

 A large amount of free nucleic acid (DNA or RNA) is present in the body fluid (plasma, serum or pleural effusion) of a tumor patient, and its content is about 10 times higher than that of a healthy person. This type of free nucleic acid is mainly derived from apoptosis and necrosis of cancer cells, and its genetic characteristics are identical to those of tumor genomic DNA. Therefore, serum free nucleic acid can be used for detection of oncogene mutation. However, free nucleic acids may have only a very small number of mutated genes, but a large number of wild-type genes, a large number of mixed wild-type genes will hinder the detection of mutant genes, thus requiring a highly sensitive and specific mutant detection method. To assess EGFR gene mutations.

According to current literature reports, the frequency of EGFR-related gene mutations is about 30%-50% in Asia, mainly in the base deletion mutation of 19 exons (ΔΕ746-750Α, including two common types, See the table below) and the point mutation of the 21 exon (L858R). In China, the base deletion mutation of 19 exons accounted for about 54.5 % of the total number of mutations, the point mutation of 21 exons accounted for about 40.3 %, and the mutations of other exons only accounted for 5.2%. To this end, the present invention selects the 19 exons and 21 exons with the highest mutation rate for detection. Exonl9 Common Missing Type Missing Length Exonl9 Sequence (one indicates missing part)

Wild type GGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAA

 GGAATTAAGAGAAGCAACATCTCCGAAAGCCAACAAGGAAATCCTCGAT

Del E746-A750 (1) 15bp GGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAA (abbreviated as E19M1) AACATCTCCGAAAGCCAACAAGGAAATCCTCGAT del E746-A750 (2) 15bp GGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAA (abbreviated as E19M2) G ACATCTCCGAAAGCCAACAAGGAAATCCTCGAT

 One of the objects of the present invention is to provide a probe sequence for the detection of mutations in the EGFR gene.

 The technical solution to achieve the above objectives is as follows:

 Probe for detection of mutations in the EGFR gene: including

 a wild-type probe selected from exonl9 exons selected from any one of SEQ ID N0.1 to SEQ IDN0.3, and an exo-expressed exonl9 selected from any one of SEQ ID N0.4 - SEQ ID N0.6 a del E746-A750 (1) mutant probe, Wo IV or a mutant del E746-A750 (2) probe selected from the exonl9 exon selected from any one of SEQ IDN 0.29 to SEQ IDN 0.31; and / or

 a wild type probe selected from the group consisting of 3 £(510^).7~86 (3101»10.9 for the £0^1 exon, and selected from any one of SEQ ID NO. 10 to SEQ ID N0.12 A mutant probe directed against the Exon21 exon.

 Another object of the present invention is to provide a liquid phase chip for detecting mutations in the EGFR gene. The liquid phase chip can be used to detect related mutations in the exons 19 and 21 of the EGFR gene, and can be used to predict the therapeutic effects of gefitinib, erlotinib and the like.

 The technical solution to achieve the above objectives is as follows:

 A liquid phase chip for detecting EGFR gene mutation, mainly comprising: a coated base sequence selected from the group consisting of SEQ ID

a microsphere of any one of N0.1 to SEQ IDN0.3 for an amino-modified probe of the exonl9 exon wild type, and a base sequence of the coating selected from SEQ ID N0.4 to SEQ ID N0.6 A globule, and/or a coated base sequence for an exonl9 exon del E746-A750C 1 ) mutant amino modified probe selected from any one of SEQ ID N0.29 to SEQ ID N0.31 Microspheres of amino modified probes against Exonl9 exon del E746-A750 (2) mutant; and/or

 a microsphere coated with an amino-modified probe for the Exon21 exon wild type of any one of SEQ IDN0.7 to SEQ ID N0.9, and a base sequence selected from the group consisting of SEQ ID a microsphere for an amino-modified probe of the Exon21 exon mutant of any one of NO. 10 to SEQ ID NO. 12, wherein a spacer arm is attached between the base sequence of each of the probes and the amino group, each of the above The microspheres have different color coding;

 And a primer for amplifying a target sequence having a 19 exon and/or 21 exon mutation site, and the end of the target sequence has a biotin label.

The spacer arm described above is used to separate a specific probe from the surface of the microsphere or to place the specific probe in the pro Sequence in an aqueous environment. By arranging a spacer arm sequence of appropriate length between the probe sequence and the amino group, steric hindrance can be reduced, the efficiency of the hybridization reaction, and the specificity of the hybridization reaction can be improved. Common spacer sequences include poly dT, poly (dT), oligotetraethylene glycol, and (CH2) n spacer arms (n ≥ 3), such as (CH2) 12, (CH2) 18 and the like. In addition, if poly (dA) interference is present, poly (TTG) can also be used as the spacer arm. The spacer arm of the present invention is preferably 5-30 T, more preferably 10 D.

 Composition of amino-modified probes with spacer arms SEQ ID NO.

 5 'NH2-TTTTTTTTTTcaaggaattaagagaagcaacat3 ' 1

5 ^, NH2-TTTTTTTTTTaaggaattaagagaagcaac3, 2

 5 'NH2-TTTTTTTTTTgttgcttctcttaattcctt3 ' 3

 5 'NH2-TTTTTTTTTTgtcgctatcaaaacatctccg3 ' 4

 5 'NH2-TTTTTTTTTTtcgctatcaa acatctccg3 ' 5

 5 'NH2-TTTTTTTTTTcggagatgttttgatagcga3 ' 6

 5 'NH2-TTTTTTTTTTagattttgggcTggccaaact3 ' 7

 5 'NH2-TTTTTTTTTTgattttgggcTggccaaact3 ' 8

 5 'NH2-TTTTTTTTTTagtttggccAgcccaaaatc3 ' 9

 5 'NH2-TTTTTTTTTTtttgggcGggccaaactctg3 ' 10

 5 'NH2-TTTTTTTTTTgattttgggcGggccaaact3 ' 11

 5 'NH2-TTTTTTTTTTagtttggccCgcccaaaatc3 ' 12

 5 'NH2-TTTTTTTTTT ccgtcgctatcaagacatctc 3' 29

 5 'NH2-TTTTTTTTTT cgtcgctatcaagacatct 3' 30

 5 'NH2-TTTTTTTTTT gagatgtcttgatagcgacgg 3' 31.

 Preferably, the primer for amplifying a target sequence having a 19-exon mutation site comprises a primer sequence of SEQ ID N0.13 to SEQ ID NO. 15, wherein at least one of the primers has a terminal biotin a marker; and/or a primer for SEQ ID N0.16 to SEQ ID N0.18 for amplifying a target sequence having a 21 exon mutation site, wherein at least one of the primers has a terminal biotin label.

 Another object of the present invention is to provide a method for detecting mutation of an EGFR gene, which is rapid, accurate, and simple in operation, and can simultaneously detect multiple mutation sites in parallel. The sample to be tested can be a tissue sample or a serum. Plasma or pleural effusion.

 A method for detecting a mutation in a liquid phase chip EGFR gene using the above EGFR gene mutation detection, comprising the following steps:

(1) after extracting the DNA in the sample to be detected, performing the first round of PCR amplification using the target substance for amplifying the target sequence having the 19 exon and/or 21 exon mutation sites;

 (2) enzymatically enriching the first round of PCR amplification products;

 (3) performing a second round of PCR amplification using the digested product as a template;

(4) the second round of PCR amplification products are hybridized with the above-mentioned microspheres coated with the probe sequence; (5) After the hybridization reaction, streptavidin-phycoerythrin was added for reaction, and then the signal was detected by a Luminex apparatus. Preferably, the first round of PCR amplification is performed as: SEQ ID NO. 13, SEQ ID NO. 14 or SEQ ID NO. 16, SEQ ID NO. 17; performing a second round of PCR amplification The pair of bows used: SEQ ID NO. 13, SEQ ID NO. 15 and/or SEQ ID N 0.18, SEQ ID N 0.17. Preferably, the temperature of the hybridization is 55-60 °C.

 The main advantages of the invention are:

 (1) The EGFR gene mutation detecting liquid phase chip provided by the invention can simultaneously detect a site with a relatively high frequency of EGFR gene mutation, and can detect 19 exons and 21 exons separately, or Simultaneous detection of both, uniform reaction conditions during detection, good detection results, high sensitivity, detection accuracy of more than 90%.

 (2) The detection method of the present invention has simple steps, thereby avoiding many uncertain factors existing in a complicated operation process, and thus the detection accuracy can be greatly improved.

 (3) The detection method provided by the present invention requires much less time than the commonly used sequencing technology, and is particularly in line with clinical needs.

 (4) The present invention adopts a method of enzymatic digestion and enrichment to carry out PCR amplification of a target sequence and is used for detection, thereby avoiding interference caused by a large number of wild-type sequences in the product.

 (5) The detection method provided by the invention not only can detect the EGFR gene mutation in the tumor tissue sample, but also can detect the EGFR gene mutation in the body fluid of the tumor patient, thereby having the advantages of convenient sampling, small patient pain, and dynamic monitoring. detailed description

 Polystyrene microspheres were used as the reaction carrier, and the fluorescence detector was used as a detection platform to perform high-throughput multi-index parallel detection of nucleic acid molecules. In the manufacturing process of the microspheres, different proportions of red and infrared light dyeing agents are incorporated to form up to 100 different color coded microspheres. Different microspheres covalently bind nucleic acid molecules for different analytes as probe molecules, reporter molecules are labeled with biotin, and stained with highly sensitive fluorescent dyes. These microspheres and the analyte, reporter, and fluorescent label form a complete microsphere detection system for reading in the Luminex system. The Luminex reading system inspires the red laser and the green laser to detect the microsphere system, respectively. The red laser detects the intensity of the red-classified fluorescence on the surface of the microsphere and numbers it according to the different colors in the microsphere to determine the type of reaction; The fluorescence intensity of the fluorescent label in the sample is detected, and the type and quantity of the microspheres detected by the laser are automatically statistically analyzed by a machine and a computer to determine the respective concentrations of the plurality of target test objects of the sample to be tested.

Solution formula:

Coupling solution (pH 4.5): O.lmol/L MES (Sigma M-2933)

Washing solution: 0.2ml/L Tween-20 (Sigma P-9416), lg/L SDS (Sigma L-4390)

TE (pH 8.0) (stock solution): lOmmol/L Tris (Sigma 337501), lmmol/L EDTA (Sigma E-5134) 2xTm hybridization buffer Reagent source final concentration per 250ml lMTris-HCl, pH 8.0 SigmaT3038 0.2M 50ml

 5M NaCl Sigma S5150 0.4M 20ml

 Triton X-100 Sigma T8787 0.16% 0.4ml

 Filtered and stored at 4 ° C

Example 1

 Preparation of liquid phase chip for detection of EGFR gene mutation

 First, probe sequence design and microsphere coating

Specific oligonucleotide probes were designed for wild-type and mutant sequences of the 19, 21 exons of the EGFR gene. The 5' end of the probe is an amino group followed by a 10 T spacer arm. The probe was synthesized by Shanghai Biotech Engineering Services Co., Ltd. The probes were coupled together by covalent bonding with different color-coded microspheres (purchased from Luminex) (coating process). The probe sequences are shown in the following table:

每种微球包被的具体步骤如下：

The specific steps for each microsphere coating are as follows:

(1) Centrifuge the probe dry powder at 10,000 rpm for 1 min; dissolve with dd3⁄40 to O. lmM (O. lnmol/μΐ, about 70 μl) _; shortly centrifuge to concentrate the solution on the bottom of the tube, dispense as ΙΟμΙ and 2μ1, store in - 20 ° C _;

 (2) taking the microsphere mother liquor, vortexing, and fully mixing into a microsphere suspension;

(3) Remove lx lO ⁵ microspheres (8 μl total mother liquor) into a 0.5 ml centrifuge tube; centrifuge at 15,000 rpm lOmin, carefully discard the supernatant;

 (4) Add ΙΟμΙ coupling solution (pH 4.5), vortex and mix well;

(5) Take 2μ1 probe mother solution and dilute to 2ριη ₀ 1/μ1 with coupling solution (ρΗ4.5) _;

(6) Add the corresponding 2ρπι ₀ 1/μ1 probe working solution 2μ1 to the centrifuge tube containing the microspheres; (7) Prepare the EDC working solution (10 mg/ml) with ddH ₂ 0 _;

 (8) Add 2.5μ1 EDC working solution to each reaction tube and discard the remaining EDC working solution;

(9) Incubate at 25 ° C for 30 min; add 2.5 μl EDC working solution to each reaction tube;

 (10) Incubate at 25 ° C for 30 min;

 (11) Add 0.2ml of washing solution, vortex and mix well, centrifuge at 12,000g for 5min, carefully discard the supernatant; repeat this step once;

 (12) Add 500μl solution (ρΗ8.0), vortex and mix well;

 (13) Centrifuge at 12,000g for 5min, carefully discard the supernatant;

(14) Add 17μl ΤΕ solution (ρΗ8.0) and vortex to mix well. (The concentration of microspheres should be approximately 5χ10 ³ /μΐ);

 (15) Take 2μ1, dilute 50 times with water, count; store at 2-8C.

 Second, the detection of the effect of the microspheres

Six groups of experiments were set up in exon 19 and exon 21, respectively. Taking the 19 exons as an example, the microspheres were tested for the effect of detecting the microspheres containing the wild type probe in the first reaction system and the corresponding complementary sequence of the biotin label for the coating effect detection, ie E19w- P+E19w-b; the second set of reaction system contains wild-type probe-coated microspheres, the mutant probe-coated microspheres and the reverse complement of the biotin-labeled wild-type probe, ie E19w-P +E19m-P+E19w-b _; the reverse complement of the mutant probe-coated microspheres and the biotinylated wild-type probe in the third reaction system is E19W-P+E19m-b; The reverse complementary sequence of the mutant probe-coated microspheres and the biotin-labeled mutant probe in the reaction system is E19m-P+E19m-b; the fifth group reaction system contains the mutant probe coated The reverse complement of the microspheres, the wild-type probe-coated microspheres and the biotin-labeled mutant probe is E19w-P +E19m-P+E19m-b _{; the} sixth set of reaction systems contains the mutant probe The reverse complement of the coated microspheres and the biotinylated wild type probe is E19m-P+E19w-b. The exon 19 microspheres were tested for effect detection experiments with the same 19 exons. The specific operation process is as follows:

(1) vortex the microsphere tube to fully mix the microspheres;

(2) Dilute the microspheres to 75 / μΐ with 1.5xTm hybridization solution (33μl of microsphere working solution per reaction) _;

 (3) vortex the microsphere working solution to make it fully mixed; add 33μ1 microsphere working solution to each well;

 (4) Dilute the reverse complement sequence of biotin-labeled probes for coating effect detection (ie E19w-b, E19m-b, E21w-b, E21m-b) to 25ήη1/17μ1 with ΤΕ (ρΗ8.0) ;

 (5) The background hole is added to 17 μl (pH 8.0);

(6) Add 17μ1 biotin-labeled probe reverse complement sequence for effect detection to each well (ie) E19w-b, E19m-b, E21w-b, E21m-b) working solution (dissolved in TE solution);

 (7) Gently mix the hook; cover the reaction plate (tube cover) to prevent evaporation;

 (8) Set up the PCR program: 95 ° C for 3 min; 60 ° C hybridization for 15 min;

 (9) Prepare SA-PE to 10ug/ml with l xTm hybrid solution; add 25μ1 SA-PE to each well and mix gently;

(10) 60 ° C hybridization incubation for 5 min;

 (11) Set the Luminex instrument to the hybridization temperature and read.

 The result of the microsphere coating effect detection is as follows

 The experimental results show that the 19 exon wild type probe, 19 exon mutant probe, 21 exon wild type probe and 21 exon mutant probe designed by the invention can be well recognized. Corresponding complementary sequences, the fluorescence values after hybridization are all above 2500. At the same time, there is no cross-reaction between the 19 exon wild type probe and the 19 exon mutant type probe; 21 exon reaction between the exon wild type probe and the 21 exon mutant type probe, However, the cross-reaction rate is within 10%, which basically does not affect the test results.

 For the gene sequences of EGFR gene 19 and 21 exons, the following primers were designed:

The primers were synthesized by Shanghai Shenggong Bioengineering Technology Co., Ltd. Among them, Exonl9 - Sl and Exonl9 - Asl are used The first round of PCR amplification of exon 19, biotin-labeled Exonl9-Sl and Exonl9-As2 were used for the second round of PCR amplification of exon 19; Exon21-S1 and Exon21-Asl were used for exon 21 For the first round of PCR amplification, Exon21-Asl and biotinylated Exon21-S2 were used for the second round of PCR amplification of exon 21. The prepared liquid phase chip for detecting EGFR gene mutation includes:

 Microspheres coated with a wild-type amino-modified probe for Exonl9 exon of SEQ ID NO. 3, coated with

Microspheres of the del E746-A750(1) mutant amino-modified probe of Exonl9 exon of SEQ ID NO. 6, coated with del E746-A750 of Exonl9 exon of SEQ ID N0.31 (2) Microspheres of a mutant amino-modified probe, microspheres coated with a wild-type amino-modified probe against Exon21 exon of SEQ ID N0.9, and coated with SEQ ID N0.12 a microsphere of a mutant amino-modified probe of the Εχοιώΐ exon, each of the above microspheres having a different color code;

 The primer sequences of SEQ ID NO: 0.13 - 15 and SEQ ID NO: 0.16-1, wherein the 5' ends of the base sequences of SEQ ID NO. 13 and SEQ ID NO. 18 are respectively labeled with biotin. Example 2

 Detection of non-small cell lung cancer serum samples using the liquid phase chip detected by the EGFR gene mutation in Example 1. Preparation of the sample to be tested (extraction of free nucleic acid in plasma, serum and pleural fluid):

 Refer to the AxyPrep Whole Blood Genome Small Extraction Kit for instructions. The detailed steps are as follows:

 (1) Take about 2.5ml of anticoagulated venous blood or pleural effusion, centrifuge for 15 minutes at 3000rpm, and add 300μ1 supernatant to a 1.5ml clean sterile centrifuge tube;

 (2) Add 500μ1 ΑΡ1 buffer to the centrifuge tube and mix well by vortexing;

 (3) Add 100μ1 ΑΡ2 buffer and mix well by vortexing;

 (4) Centrifuge at 12, OOO rpm for 10 minutes at room temperature;

 (5) Carefully pipette the supernatant into the adsorption column AxyPrep placed on a 2ml collection tube, cover, and centrifuge at 6, OOOrpm for 1 minute;

 (6) The waste liquid in the waste collection tube is drained, washed once with 800 μl buffer W1, and centrifuged at 6, OOO rpm for 1 minute;

(7) Drain the waste liquid from the waste collection tube, add 800 μl buffer W2, and centrifuge at 12, OOO rpm for 1 minute; pour off the waste liquid from the waste collection tube, add 500 μl buffer W2, and centrifuge at 12, OOO rpm. 1 minute, discard the waste liquid;

(8) Put the adsorption column AxyPrep back into the empty collection tube and centrifuge at 12, OOO rpm for 1 minute;

 (9) Place the adsorption column AxyPrep in a clean and sterile 1.5ml centrifuge tube, add 40μl buffer, leave it at room temperature for 1 minute, centrifuge at 12, OOOrpm for 1 minute, elute the DNA, check by electrophoresis, store at -20 °C. .

2. PCR amplification and enzymatic digestion of the sample to be tested:

(1) PCR amplification and enzymatic digestion of sample DNA Enzyme-enriched PCR amplification of exon 19 mutants is as follows -

1. The first round of PCR amplification

 PCR reaction system:

 10 X TureStart DNA polymerase Buffer 2.5 μΐ

 2.5mMdNTP mixture 2μ1

 Εχοη 19-S1 0.5μ1 (20μιηο1/ί)

Εχοη 19-Asl 0.5μ1 (20μπιο1/ί)

TureStart Taq DNA polymerase 0.5μ1 (21Ι/μ1 ) Template DNA Ιμΐ Add sterile double distilled water to 25μ1

PCR reaction conditions:

 Step Temperature Time Cycle number Pre-denaturation 95 °C 5 min 1 Denaturation 95 V 30 sec

 Refolding 60 °C 30 sec 30 extension 72 . C 45 sec

 Finally extended 72. C 10 min 1 PCR product Mse l digestion:

The reaction system is as follows:

 10χ Mse l Buffer Ιμΐ

 PCR product 3μ1

 lOOxBSA 0.1 μΐ

 Mse I 0.5μ1 ( 50υ/μ1) Add sterilized double distilled water to ΙΟμΙ

 The enzyme was incubated at 37 ° C for 2 hours and at 65 ° C for 20 minutes.

Second round of PCR amplification

PCR reaction system:

 lOxTureStart DNA polymerase Buffer 2.5μ1 2.5mMdNTP mixture 2μ1

 Biotin-Exon 19-S1 0.5μ1 ( 20μηιο1/ί)

Εχοη 19 -As2 0.5μ1 ( 20μη ο1/ί )

TureStart Taq DNA polymerase 0.5μ1 ( 21Ι/μ1) Digested product Ιμΐ

 Add sterile double distilled water to 25μ1

 PCR reaction conditions:

 Step Temperature Time Cycle number Pre-denaturation 95 °C 5 min 1 Denaturation 95 °C 30 sec Refolding 60 °C 30 sec 30 Extension 72. C 45 sec

 Final extension 72 °C 10 min 1 exon 21 mutant enzymatic digestion enrichment PCR amplification is as follows:

 First round PCR amplification

 PCR reaction system:

 lOxTureStart DNA polymerase Buffer 2.5μ1

 2.5mMdNTP mixture 2μ1

Exon21-Sl 0.25μ1 (20μηιο1/ί)

Exon21-Asl 0.25μ1 (20μηιο1/ί)

TureStart Taq DNA polymerase 0.5μ1 (2υ/μ1) Template DNA Ιμΐ Add sterile double distilled water to 25μ1 PCR reaction conditions:

 Step Temperature Time Cycle number Pre-denaturation 95 °C 5 min 1 Denaturation 95 °C 30 sec Refolding 60 °C 30 sec 30 Extension 72 °C 45 sec Last extension 72. C 10 min 1

 2. PCR product Mscl digestion:

 The reaction system is as follows:

 10χ Mscl Buffer Ιμΐ PCR product 3 1 Msc I 0.5μ1 (lOU/μΙ) plus enzyme-free water to ΙΟμΙ

Incubation at 37 °C for 2 hours, 65 °C for 20 minutes inactivation of enzymes - 3. Second round of PCR amplification

 PCR reaction system:

 lOxTureStart DNA polymerase Buffer 2.5μ1

 2.5mMdNTP mixture Ιμΐ

 Biotin-Exon 21 -S2 0.5μ1 (20μπιο1/Ε)

 Exon 21-Asl 0.5μ1 (20μπιο1/ί)

 TureStart Taq DNA polymerase 0.5μ1 (2υ/μ1 )

 Digested product Ιμΐ

 Add sterile double distilled water to 25μ1

 PCR reaction conditions:

 Temperature time cycle number

 Pre-denaturation 95. C 5 min 1

 Denature 95. C 30 sec

 Refolding 60 V 30 sec 30

 Extend 72 V 45 sec

 Last extension 72 °C 10 min 1

Or simultaneous amplification of the EGFR gene 19, 21 exon mutant and wild-type sequence (multiplex PCR amplification)

1. 19 exons, 21 exons The first round of PCR amplification and digestion is the same as above, except that the second round of PCR amplification adopts simultaneous amplification.

 2. The second round of PCR amplification (19, 21 exon wild type sequence and mutant sequence simultaneous amplification)

 PCR reaction system:

 lOxTureStart DNA polymerase Buffer 2.5μ1

 2.5mMdNTP mixture 2μ1

 Biotin-Exon 19—SI 0.5μ1 (20μπιο1/Ε)

 Exon 19 -As2 0.5μ1 (20μπιο1/

 Biotin-Exon 21 -S2 0.5μ1 (20μπιο1/

 Exon 21-Asl 0.5μ1 (20μιηο1/

 TureStart Taq DNA polymerase Ιμΐ (2υ/μ1)

 19 exon digestion product Ιμΐ

 21 exon digestion product Ιμΐ

 Add sterile double distilled water to 25μ1

PCR reaction conditions: Step temperature time cycle

 Pre-denaturation

 95 °C 5 min 1

 Denaturation 95 Ό 30 sec

 Refolding 60 °C 30 sec 30

 Extend 72 °C 45 sec

 Final extension 72 °C 10 min 1 III. Hybridization and on-machine detection

 (1) will be coated with 19 exon wild type probe (E19w-P), 19 exon del E746-A750 (l) mutant probe (E19m-P), 19 exon del E746-A750 (2) Mutant probe (E19m-Pll), 21 exon wild type probe (E21w-P), 21 exon mutant probe (E21m-P), negative control probe (NP) micro The ball is vortex 30s, sonicated for 30s;

 (2) using 1.5xTm hybridization solution to prepare a mixed microsphere working solution containing a probe, so that the concentration of each microsphere in the solution is 75 / μΐ;

 (3) The mixed microsphere working fluid will be vortex 30s, sonicated for 30s;

 (4) Add the corresponding 33 μM mixed microsphere working solution to each well, so that the reaction system finally contains about 2500 each of the microspheres;

(5) Add Πμΐ TE (pH 8.0) to the background well; add 2-17μ1 of each of the 19 exons and 21 exons of each sample to each well, add TE to 17μ1; Lightly mix; cover the reaction plate (tube cover) to prevent evaporation;

(6) Set the PCR program: 95 ° C 5 min; 60 ° C hybridization incubation for 15 min;

 (7) Prepare SA-PE to 2 ug/ml (75 μl/well) with 1 x Tm hybridization solution;

 (8) Centrifuge the reaction tube (plate) at 12,000 g for 5 min, and carefully discard the supernatant. Or transfer to the filter plate and filter to remove the liquid;

 (9) Add 75μ1 SA-PE (streptavidin phycoerythrin) working solution (150ng SA-PE) to each well and mix gently;

 (10) Immediately placed at the hybridization temperature (60 ° C), incubated for 5 min;

 (11) Set the Luminex instrument to the hybridization temperature and read.

Fourth, test results and data analysis

The product after the reaction was detected by a Luminex series analytical instrument, and the test results are shown in Table 1. There is no cross-reactivity between the 19 exon wild type and the mutant (deletion) probe, so the mutant fluorescence value (the higher of the two mutant fluorescence values) is divided by the negative control fluorescence value (ie M/N) > 25 As a positive judgment value (cut-off value). When the 19 exon detection result M/N>25, it was determined that the sample was absent in the presence of exon 19, otherwise the sample was determined to be wild type of exon 19. There is a cross-reaction between the 21 exon wild type probe and the mutant probe (the cross-reaction rate is within 10%), therefore, (mutative fluorescence value - negative control fluorescence value) / (wild type fluorescence value - negative control fluorescence value) SP ((MN) / (WN)) > 2 was used as a positive judgment value (cut-off value). When the 21 exon detection result ((MN) I (WN) ) 〉 2, it was determined that there was a L858R point mutation of 21 exon in the sample, otherwise the sample was judged to be wild type of exon 21 .

 According to this criterion, among the 10 samples detected in this example, 3 cases had deletions of 19 exons (samples No. 3, No. 5, No. 7), and 2 cases had point mutations of 21 exons (2). No., sample 10). The results were compared with the results of traditional polyacrylamide gel electrophoresis followed by silver staining to calculate the coincidence rate. The detection coincidence rate of exon 19 reached 100%, and the detection coincidence rate of exon 21 also reached 90%. The overall anastomotic rate was 95%. Therefore, the EGFR gene mutation detection liquid phase chip and the detection method thereof provided by the invention can accurately detect the deletion mutation of the exon 19 of the EGFR gene and the point mutation of the exon 21, and the accuracy rate is as high as 95%. The effectiveness of targeted drugs such as fentanib and erlotinib provides an important means of testing for clinically accurate drug use, avoiding aging loss of unnecessary treatment and economic loss.

 Table 1 Test results of serum samples

表 2 血清样本 EGFR突变类型分析结果

Table 2 Results of EGFR mutation type analysis of serum samples

3 177.06 0.08 19外显子缺失 19外显子缺失

3 177.06 0.08 19 exon deletion 19 exon deletion

4 5.71 1.10 wild type wild type

 5 469.13 0.12 19 exon deletion 19 exon deletion

6 5.14 0.75 wild type wild type

 7 95.68 1.90 19 exon deletion 19 exon deletion

8 22.75 0.81 wild type wild type

 9 0.60 1.18 wild type wild type

 10 4.22 2.26 21 exon point mutation wild type Example 3

 Detection of lung cancer tissue samples by liquid phase chip using EGFR gene mutation detection in Example 1

 The lung cancer tissue samples of the patient No. 1-10 used in the above Example 2 were tested, and the specific process is as follows: 1. Preparation of the sample to be tested

Extraction of _DNA from lung cancer tissue samples: Take 5-50 mg of tissue specimens after lung cancer biopsy or biopsy. After grinding, wash twice with PBS solution of pH 7.4; Wash the tissue specimens and resuspend in 1 ml of digestive juice (50 mmol/ L Tris, lmmo/L Na ₂ EDTA, 0.5% Tween-20, 200 ug/ml proteinase K 200, pH 8.5), digested in a 55 ° C water bath for 1 hour, 99 ° C water bath for 15 min inactivated proteinase K; 12000 rpm The mixture was centrifuged for 10 minutes; the supernatant was taken, and extracted by a phenol-chloroform-isoamyl alcohol method, and a DNA sample for a PCR reaction was obtained by an ethanol precipitation method. DNA can also be extracted by microcentrifugation column method; 2. PCR amplification and enzymatic digestion of the sample to be tested:

 The specific method is the same as in the second embodiment.

Third, hybridization and machine testing:

 The specific method was examined in the same manner as in Example 2.

Fourth, test results and data analysis

The products after the reaction were detected by a Luminex series analytical instrument, and the results are shown in Table 3 and Table 4. The criteria for determination are as described in Example 2. The experimental results show that the results of the analysis of lung cancer tissue samples are consistent with the results of the analysis using serum samples, that is, there are 3 cases of 19 exon deletion mutations in the 10 samples (samples No. 3, No. 5, No. 7), 2 cases of 21 exon mutations (No. 2, sample No. 10). The method for detecting EGFR gene mutation using serum free nucleic acid provided by the present invention is feasible, and the method provided by the present invention is stable and reliable. Table 3 Test results of lung cancer tissue samples

Table 4 Results of EGFR mutation type analysis of lung cancer tissue samples

 Example 4

1. Preparation of liquid phase chip for detecting EGFR gene mutation EGFR mutation detection for other alternative probe designs and microsphere coatings:

 The probe sequences are shown in the following table:

 Two sets of oligonucleotide probes (E19w-Pl, E19m-Pl, E19m-P13, E21w-Pl, E21m-Pl and E19w-P2) were designed for the wild type and mutant sequences of the EGFR gene 19 and 21 exons. , E19m-P2, E19m-P12,

E21W-P2, E21m-P2) The 5, end of the ₀ probe is an amino group, followed by a 10 T spacer arm. The probe was synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd. The probes were coupled together with co-valent binding to different color-coded microspheres (available from Luminex Corporation), respectively, as described in Example 1.

 The liquid phase chip for detecting EGFR gene mutation in this embodiment includes:

Microspheres coated with a wild-type amino-modified probe for Exonl9 exon of SEQ ID NO. 2, a mutant amino-modified with SEQ IDN0.5 for Exonl9 exon delE746-A750 (1) Microspheres of the probe, microspheres coated with a mutant amino-modified probe of SEQ ID NO. 30 against Exonl9 exon del E746-A750 (2), coated with Exon21 outside SEQ ID N0.8 Microspheres of a wild-type amino-modified probe of the exon, and microspheres of a mutant amino-modified probe coated with Exon21 exon of SEQ ID NO. 11 , each of the above microspheres Have different color coding; and

 The primer sequences of SEQ ID NO. 13-15 and SEQ ID NO. 16-18, wherein the 5' end of the base sequence of SEQ ID NO. 15 and SEQ ID N 0.17 is added with a biotin label, respectively.

 2. The liquid phase chip for detecting EGFR gene mutation of the present embodiment is used for detecting lung cancer serum samples

 Detection of non-small cell lung cancer serum samples using E19w-Pl, E19m-Pl, E19m-P13, E21w-Pl, E21m-Pl probe-coated microspheres.

 Five to five non-small cell lung cancer serum samples used in Example 2 were selected for detection. The preparation of the sample to be tested, the PCR amplification and enzymatic digestion of the sample to be tested, the experimental steps of hybridization and on-machine detection are the same as those described in Example 2. The experimental results are as follows:

 Table 5 Test results of serum samples

表 6血清样本 EGFR突变类型分析结果

Table 6 results of EGFR mutation type analysis of serum samples

 The experimental results show that the probes E19W-P1' E19m-Pl, E19m-P13, E21w-Pl, E21m-Pl can also be used for mutation detection of EGFR in clinical samples, and the results of analysis and probes (E19w-P, E19m-P) , E19m-Pll, E21w-P, E21m-P) The results were consistent. Example 5

 1. Preparation of liquid phase chip for detecting EGFR gene mutation

 Preparation method is the same as the embodiment 1.

 The liquid phase chip for detecting EGFR gene mutation in this embodiment includes:

Microspheres coated with a wild-type amino-modified probe of Exonl9 exon having SEQ ID NO. 1, coated with Microspheres of the mutated amino-modified probe of SEQ ID NO. 4 against E _XOn 19 del E746-A750 ( 1 ), coated with SEQ ID N0.29, for Exonl9 exon del E746-A750 (2 Microspheres of a mutant amino-modified probe, microspheres coated with a wild-type amino-modified probe against Exon21 exon of SEQ ID N0.7, and Exon21 coated with SEQ ID NO. Microspheres of a mutant amino-modified probe of an exon, each of which has a different color coding;

 The primer sequences of SEQ ID NO: 0.13-15 and SEQ ID NO. 16-18, wherein the 5' ends of the base sequences of SEQ ID NO. 15 and SEQ ID NO. 17 are each added with a biotin label.

 2. The liquid phase chip for detecting EGFR gene mutation in the present embodiment is used for detecting serum samples of lung cancer using E19w-P2, E19m-P2, E19mP12, E21w-P2, E21m-P2 probe coated microspheres for non-small Detection of serum samples of cell lung cancer.

 A total of 1-5 serum samples from five non-small cell lung cancers were also selected for testing. The preparation of the sample to be tested, the PCR amplification and enzymatic digestion of the sample to be tested, the experimental steps of hybridization and on-machine detection are the same as those described in Example 2. The experimental results are as follows: Table 7 Test results of serum samples

表 8血清样本 EGFR突变类型分析结果

Table 8 results of EGFR mutation type analysis of serum samples

 The experimental results show that the probes E19w-P2, E19m-P2, E19m-P12, E21w-P2, E21m-P2 can also be used for the detection of mutations in clinical samples of EGFR, and the results of analysis and probes (E19w-P, E19m-P, E19m-Pl l , E21w-P, E21m-P) The results were consistent.

Claims

Rights request  1. A probe sequence for detecting an EGFR gene mutation, which is characterized by:  a wild type probe selected from any one of SEQ ID N0.1 to SEQ ID N0.3 for Exonl9 exon, and selected from any one of SEQ ID N0.4 to SEQ ID N0.6, Del E746-A750(l) mutant probe for Exonl9 exon, and/or del E746-A750 for exonl9 exon selected from any one of SEQ ID N0.29~SEQ ID N0.31 (2) a mutant probe; and/or  a wild type probe selected from the exon 21 exon of any one of SEQ ID N0.7 to SEQ ID N0.9, and selected from any one of SEQ ID NO. 10 to SEQ ID NO. A mutant probe directed against the Exon21 exon.  2. A liquid phase chip for detecting EGFR gene mutation, which is characterized by: A. Microspheres coated with a probe: an amino-modified probe having a base sequence selected from any one of SEQ ID N0.1 to SEQ ID N0.3 for wild type of Εχ _0η 19 exon Microspheres, and amino-modified probes having a base sequence selected from the group consisting of SEQ ID N0.4 - SEQ ID N0.6 for the Exonl9 exon del E746-A750(l) mutant Microspheres, and/or amino-modified probes having a base sequence selected from any one of SEQ ID N0.29 to SEQ ID N0.31 for Exonl9 exon del E746-A750(2) mutant Microspheres; and/or  a microsphere coated with an amino-modified probe for the Exon21 exon wild type of any one of SEQ ID N0.7 to SEQ ID N0.9, and the base sequence of the coating is selected from a microsphere for the amino-modified probe of the Exon21 exon mutant of any one of SEQ ID NO. 10 to SEQ ID N0.12, wherein a spacer arm is connected between the base sequence and the amino group of each of the above probes, Each microsphere has a different color coding;  B. Primer: A primer for amplifying a target sequence having a 19 exon and/or 21 exon mutation site, and the end of the target sequence has a biotin label.  The liquid phase chip for detecting EGFR gene mutation according to claim 2, wherein the probe-coated microspheres are: coated with a base sequence of SEQ ID N0.3, and exposed to Exonl9 Microspheres of a sub-wild type amino-modified probe, and microspheres coated with an amino-modified probe having a base sequence of SEQ ID N0.6 against an Exonl9 exon mutant, and a coated base sequence a microsphere of SEQ ID N0.31 for an amino-modified probe of the Exonl9 exon mutant;  Microspheres coated with an amino-modified probe for the Exon21 exon wild type with the base sequence of SEQ ID N0.9, and a base sequence of SEQ ID N0.12 for the Exon21 exon mutant The microspheres of the amino-modified probe have a spacer arm interposed between the base sequence of each of the probes and the amino group, and each of the above microspheres has a different color code. 4. The liquid phase chip for detecting EGFR gene mutation according to claim 2, wherein: the microspheres coated with the probe are: Microspheres coated with a wild-type amino-modified probe of Exonl9 exon of SEQ ID NO. 2, micro-coated with a mutant amino-modified probe of Exonl9 exon SEQ ID N0.5 a sphere, a microsphere coated with a mutant amino-modified probe of Exonl9 exon of SEQ ID NO. 30, and a wild-type amino-modified probe coated with Exon21 exon of SEQ ID N0.8 microspheres and coated with SEQ ID NO.11 mutant amino Εχ ₀ η21 for exon probes modified microspheres, each microsphere having the above-described color-coded. The liquid phase chip for detecting mutation of an EGFR gene according to claim 4, wherein the spacer arm is 5 to 30 butyl. The liquid phase chip for detecting EGFR gene mutation according to any one of claims 2 to 5, which is characterized in that SEQ ID NO is used to amplify a target sequence having a 19 exon mutation site. 13 to SEQ ID N0.15 primer, wherein at least one of the primers has a biotin tag with a terminal; and/or SEQ ID N0.16~ for amplifying a target sequence having a 21 exon mutation site SEQ ID NO. 18 primer, at least one of which has a terminal biotin label  A method for detecting a mutation in an EGFR gene, comprising: using the liquid phase chip for detecting EGFR gene mutation according to claim 2, comprising the steps of:  1) extracting DNA in the sample to be detected for amplification of the target sequence having the 19 exon and / or 21 exon mutation sites for the first round of PCR amplification;  2) enzymatically enriching the first round of PCR amplification products;  3) performing a second round of PCR amplification using the digested product as a template;  4) the second round of PCR amplification product is hybridized with the probe-coated microspheres according to claim 2; 5) After the hybridization reaction, streptavidin-phycoerythrin is added to carry out a reaction, and then the signal is detected.  The method for detecting EGFR gene mutation according to claim 7, wherein: step 1) the primer pair for the first round of PCR amplification is: SEQ ID N0.13, SEQ ID N0.14 and/or SEQ ID N0.16, SEQ ID N0.17; Step 3) The second round of PCR amplification is: SEQ ID NO. 13, SEQ ID NO. 15, and/or SEQ ID NO. 18, SEQ ID N0.17.  The method for detecting EGFR gene mutation according to claim 7, wherein: step 4) the hybridization temperature is 55 to 60 °C.  10. The method for detecting mutation of an EGFR gene according to claim 7, wherein: the method for preparing each of the microspheres coated with the probe comprises the following steps:  (1) taking the microsphere mother liquor and mixing well into a microsphere suspension; (2) taking 8 μl of the microsphere mother liquor, containing 0.8χ 10 ⁵ —1.2χ 10 ⁵ microspheres into a 0.5 ml centrifuge tube;  (3) Centrifuge at 15,000 rpm for 10 min, discard the supernatant; add ΙΟμΙ coupling solution, vortex to mix well;  Add 2ρηιο1/μ1 probe working solution 2μ1;  (4) Add 2·5μ1 10mg/ml EDC working solution, incubate at 25°C for 30min; repeat this step once;  (5) Force to inject 0.2ml of washing solution, vortex and mix well, centrifuge at 12,000g for 5min, discard the supernatant; repeat this step once;  (6) Add 500 μl of hydrazine solution, vortex and mix well; centrifuge at 12,000 g for 5 min, discard the supernatant; (7) Add ΠμΙΤΕ solution, vortex and mix well, the concentration of microspheres should be about 5×10 ³ /μΐ;  (8) Take 2μ1, dilute 50 times with water, count, and store at 2-8'C.