CN116606913B - Hydrophilic modifier, human peripheral blood rare cell EGFR gene amplification detection kit and method - Google Patents

Hydrophilic modifier, human peripheral blood rare cell EGFR gene amplification detection kit and method Download PDF

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CN116606913B
CN116606913B CN202310785341.3A CN202310785341A CN116606913B CN 116606913 B CN116606913 B CN 116606913B CN 202310785341 A CN202310785341 A CN 202310785341A CN 116606913 B CN116606913 B CN 116606913B
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CN116606913A (en
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许嘉森
吴诗扬
刘芳
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Surexam Bio Tech Co Ltd
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Abstract

The invention provides a hydrophilic modifier, which comprises a solvent and a solute, wherein the solute comprises 0.03-10% (w/v) of carboxyethyl cellulose, 0.05-5% (w/v) of betaine, 0.05-1% (v/v) of surfactant and 0.02-0.2% (v/v) of polylysine. The invention also provides a kit for detecting EGFR gene amplification of the human peripheral blood rare cells comprising the hydrophilic modifier and a detection method using the kit. The detection kit and the detection method can improve the filtration rate of the white blood cells, prevent the cells from being stacked on the filter membrane in a large amount, prevent the cells from falling off the filter membrane easily, avoid the problems of poor imaging effect and high fluorescent background in the subsequent FISH detection, ensure the stability of the FISH detection, and well realize noninvasive real-time accurate monitoring of the EGFR gene amplification state.

Description

Hydrophilic modifier, human peripheral blood rare cell EGFR gene amplification detection kit and method
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a hydrophilic modifier, a human peripheral blood rare cell EGFR gene amplification detection kit and a detection method.
Background
The Epidermal Growth Factor Receptor (EGFR), a member of the human epidermal growth factor receptor (HER) family, also known as HER1, belongs to the family of receptor tyrosine kinases, whose genes are located on human chromosome 7. EGFR is the junction of a variety of signaling pathways, the downstream signaling pathways of which are mainly the Ras/Raf/MEK/ERK/MAPK pathway, the PI3K/PDK1/Akt pathway, the PLC-gamma pathway, the JAK/STAT pathway, and the like (Sebastin S et al Biochim Biophys Acta,2006, 1766:120-39). EGFR can participate in tumor cell proliferation, angiogenesis, invasion, metastasis, apoptosis inhibition and the like through the signal channels, and plays an important role in tumorigenesis and development. Alterations in EGFR, including EGFR gene amplification, are associated with pathogenesis and progression of many malignant tumors.
EGFR Gene amplification has been shown to occur in various tumor types (Normanno N et al, gene,2006, 366:2-16). EGFR is a common driver in lung Cancer, and the incidence of gene amplification varies from 12% to 79% (Cheng L et al, future Oncol,2011,7:519-41; tang X et al, cancer Prev Res (Phila), 2008, 1:192-200). It has been found that normal lung epithelium generally does not exhibit EGFR gene amplification or has a very low incidence of EGFR gene amplification, whereas lung Cancer EGFR gene amplification occurs at a significant elevation of 64% to 79% (Tang X et al, cancer Prev Res (Phila), 2008,1:192-200; jia XF et al, genet Mol Res,2015, 14:11006-12). Meanwhile, studies have found that the incidence of EGFR gene copy number abnormalities increases as lung cancer precancerous lesions progress (McIntire MG et al, am J Transl Res,2010,2:309-15;Sousa V et al.Virchows Arch,2011,458:571-81). These studies suggest that EGFR gene amplification may be an early event in lung cancer.
In radical surgery lung Cancer studies, it was found that the time to brain metastasis of primary tumors with EGFR gene amplification after surgery was minimal (Menghong S et al, cancer Res, 68:2163), EGFR gene amplification often meant that the overall survival of post-surgery patients was short (Kowalczuk O et al, adv Med Sci,2015, 60:277-86.) EGFR gene amplification was associated with a short disease-free survival, overall survival of post-surgery adjuvant chemotherapy patients (Koh Y et al, jpn J Clin Oncol,2011, 41:548-54), suggesting EGFR gene amplification might be a potential predictive biomarker for recurrence and/or risk of mortality after radical surgery in lung Cancer patients. In EGFR Tyrosine Kinase Inhibitor (TKI) treatment lung cancer studies, EGFR Gene amplification was found to be associated with higher therapeutic response rates, longer progression times and better survival of patients (Normanno N et al, gene,2006, 366:2-16), suggesting that EGFR Gene amplification may be a potential biomarker for efficacy assessment of lung cancer EGFR-TKI treatment.
In colorectal cancer studies, it was found that the incidence of EGFR gene amplification in the colorectal cancer population was 1.1% and in the metastatic colorectal cancer population was 4.1%, EGFR gene amplification was significantly correlated with the left primary tumor, RAS/BRAF wild type status, all tumors of the EGFR gene amplification type were microsatellite stable and HER2 non-amplified, the total survival of EGFR gene amplified metastatic colorectal cancer patients was significantly longer than EGFR gene non-amplified patients, EGFR gene amplification was an independent positive prognostic factor for metastatic colorectal cancer patients, EGFR gene amplification was significantly correlated with better total survival of RAS/BRAF wild type metastatic colorectal cancer patients receiving anti-EGFR treatment (Randon G et al J Natl Cancer Inst,2021, 113:1561-1569), suggesting that EGFR gene amplification might be a potential biomarker for assessing metastatic colorectal cancer prognosis and its anti-EGFR treatment clinical outcome.
In Gastric Cancer studies, it was found that the incidence of EGFR gene copy number gain and EGFR gene amplification in Gastric Cancer patients was 23% and 15%, respectively, EGFR gene copy number gain was more frequent in patients with lymphoid or venous invasion, advanced tumor depth, advanced stage, lymph node metastasis, HER2 positive and adjuvant chemotherapy, prognosis of EGFR gene copy number gain patients was significantly worse than patients without EGFR gene copy number gain, EGFR gene amplification was the strongest prognostic marker of the subset of EGFR gene copy number gain patients, total survival of EGFR gene gain-assisted EGFR gene amplification patients was significantly worse than EGFR gene copy number gain-assisted EGFR gene amplification patients, total survival of EGFR gene gain-assisted EGFR gene amplification patients was significantly worse than patients without EGFR gene amplification, EGFR gene copy number gain-assisted EGFR gene amplification, EGFR gene amplification-assisted EGFR gene amplification and EGFR gene amplification were both independently related to poor prognosis (higase et al, game Cancer, 19:63-73), suggesting that EGFR gene amplification was a potential prognostic strategy for detecting potential prognosis patients.
In triple negative breast cancer studies, high EGFR gene copy numbers were found to be detectable in 33% of patients, including 2% EGFR gene amplification and 31% EGFR gene polyploidy, with high EGFR gene copy numbers associated with EGFR protein overexpression, disease-free survival in high EGFR gene copy number patients being significantly shorter than in low EGFR gene copy number patients, high EGFR gene copy numbers being an independent prognostic factor for poor disease-free survival in triple negative breast cancer patients (Park HS et al, mod Pathol,2014, 27:1212-22). In breast invasive ductal carcinoma studies, EGFR gene amplification was found to be significantly correlated with ER expression, local recurrence, distant metastasis in 25% of patients, and with patient poor disease-free survival and total survival, suggesting that EGFR gene amplification may be a potential predictor of poor prognosis for breast invasive ductal carcinoma (Guo P et al, oncol Lett,2017, 14:6562-6570). These studies suggest that EGFR gene amplification detection may be a potential means of predicting prognosis of breast cancer.
The most common method for EGFR gene amplification detection at present is Fluorescence In Situ Hybridization (FISH), and the basic principle is that fluorescent signals are distinguished and counted under a fluorescent microscope through in situ hybridization of a fluorescent marked DNA probe and DNA of a sample to be detected, so that detection and diagnosis are carried out on chromosome and gene abnormal cell/tissue samples. However, FISH technology for detecting EGFR gene amplification has certain limitations in clinical application:
(1) the sample to be tested is usually a tissue sample, the material is invasive, the problems that the tissue sample is difficult to obtain or cannot obtain repeatedly, and the like exist, and the real-time monitoring of the EGFR gene amplification state cannot be realized;
(2) in some documents, rare cells in various biological fluid samples such as sputum (Kang JU et al, cancer Genet Cytogenet,2008, 184:31-7), peripheral blood (Katz RL et al, clin Cancer Res,2010, 16:3976-87), chest water (Gu Y et al, chip Med J (Engl), 2015, 128:305-9) and the like are taken as samples to be tested, usually, the cells are fixed on a glass slide and then subjected to pretreatment, enzyme digestion, in situ hybridization, washing, reading and the like, the operation steps are complex, the detection process has no unified standard operation procedure, and the fluorescence background is high;
(3) FISH is carried out on cells on a glass slide, cell preparation is critical, poor operation can cause cell overlapping, count of nuclear fluorescence signals in subsequent FISH detection is affected, and false negative results are caused;
(4) the problem of FISH on the glass slide is that the hybridization effect and the effective maintenance of cells are well balanced and the stability is kept high, the insufficient pretreatment or enzyme digestion of the cell sample may be unfavorable for the combination of the probe and the sample DNA, and further the hybridization effect is poor or even the hybridization fails, and the pretreatment or enzyme digestion of the cell sample may cause a certain damage to the cell structure or even the unclear identification or disappearance of the cell nucleus, and may cause a certain degree of cell flaking.
Disclosure of Invention
Based on the detection kit and the detection method, the detection method is simple and time-saving in operation, low in fluorescent background and good in hybridization effect, and can well realize noninvasive real-time accurate monitoring of EGFR gene amplification state.
The technical scheme for achieving the purpose comprises the following steps.
In a first aspect of the invention, a hydrophilic modifier is provided. The hydrophilic modifier comprises a solvent and a solute, wherein the solvent is water or buffer solution such as sodium acetate and sodium citrate, the solute comprises 0.03-10% (w/v) of carboxyethyl cellulose, 0.05-5% (w/v) of betaine, 0.05-1% (v/v) of surfactant, 0.02-0.2% (v/v) of polylysine, and the pH value is 6.0-8.0.
In some of these embodiments, the solute comprises 0.1% to 5 (w/v) carboxyethylcellulose, 0.05% to 3% (w/v) betaine, 0.1% to 0.8% (v/v) surfactant, 0.05% to 0.15% (v/v) polylysine, and a pH of 6.0 to 7.5.
In some of these embodiments, the surfactant is selected from any one of Triton X-100, tween20, tween80, S10.
In some of these embodiments, the further hydrophilic modifier comprises 0.01% to 0.1% (v/v) preservative. Preferably, the preservative is ProClin 300.
In some of these embodiments, the solvent for the hydrophilic modifier is 10mM sodium citrate buffer, pH 6.0.
In some of these embodiments, the solute comprises: 0.2 to 0.8 percent (w/v) of carboxyethyl cellulose, 0.08 to 0.15 percent (w/v) of betaine, 0.2 to 0.7 percent (v/v) of surfactant, 0.08 to 0.12 percent (v/v) of polylysine and the pH value is between 6.7 and 6.9.
In a second aspect, the invention provides a kit for detecting EGFR gene amplification of human peripheral blood rare cells, which mainly comprises the following components: erythrocyte lysate, any hydrophilic modifier and pretreatment liquid.
In some of these embodiments, fixatives, filters, digestive enzymes, EGFR/CEN7 probe solutions, counterstains are also included.
Further, the kit may further comprise the following other components: PBS, 4% formaldehyde solution, absolute ethanol, 70% ethanol, 85% ethanol, wash solution, and the like.
In some embodiments, the pretreatment liquid comprises 25-100 mM alkaline solution and 0.3% -1% (v/v) surfactant.
In some embodiments, the pretreatment solution comprises 25-100 mM alkaline solution and 0.3% -1% (v/v) surfactant, wherein the alkaline solution is 25-100 mM NaOH or KOH solution, and the surfactant is selected from any one of Triton X-100, tween20, tween80 and NP 40.
In some preferred embodiments, the pretreatment solution comprises 45-55 mM alkaline solution and 0.3% -0.6% (v/v) surfactant.
In some preferred embodiments, the alkaline solution is 45-55 mM NaOH solution or KOH solution; and/or the surfactant is any one of Triton X-100, tween20, tween80 and NP 40. In this case, the detection effect is better, and when the components of the pretreatment liquid are the same, the detection effect is better than that of the pretreatment liquid composed of other alkaline solutions and/or other surfactants with the same concentration.
In a third aspect, the invention provides a rare cell enrichment method for EGFR gene amplification detection of human peripheral blood rare cells.
The rare cell enrichment method for EGFR gene amplification detection of human peripheral blood rare cells comprises the following steps: s1, obtaining a human peripheral blood sample, and lysing erythrocytes to obtain a cell suspension sample from which erythrocytes are removed;
s2, filtering by a filter membrane; obtaining a pretreated cell filter membrane sample;
the step S2, the filter membrane filtration mainly comprises the following steps: (1) Performing hydrophilic treatment on the filter membrane in the filter by using any hydrophilic modifier; (2) Transferring the cell suspension sample after removing the red blood cells into a filter for filtering with a filter membrane to remove the rare cells enriched in white blood cells; (3) adding formaldehyde solution, fixing at room temperature, and removing liquid; (4) And adding PBS, washing and soaking for three times, removing liquid, and taking out the filter membrane from the filter to obtain a cell filter membrane sample.
In some of these embodiments, the hydrophilic treatment is specifically as follows: 200 mu l-600 mu l of hydrophilic modifier is added into the filter membrane of the filter, the filter membrane is soaked for 3-10 minutes at room temperature, all liquid is removed by filtration, and the filter membrane is dried at 50+/-1 ℃.
In some preferred embodiments thereof, the hydrophilic treatment is as follows: adding 350-450 mu l of hydrophilic modifier into the filter membrane of the filter, soaking the filter membrane for 5+/-0.5 minutes at room temperature, filtering to remove all liquid, and drying at 50+/-1 ℃.
The inventor of the invention researches and discovers that the filter membrane in the filter has good hydrophilicity and biocompatibility after hydrophilic treatment, and the filter membrane filtering of the cell suspension sample after removing red blood cells can improve the filtering rate of white blood cells, and is beneficial to preventing a large number of stacking phenomena of cells trapped on the filter membrane, so that the follow-up FISH detection imaging effect is avoided, and the fluorescence background is high; meanwhile, rare cells trapped on the filter membrane are not easy to fall off from the membrane in the subsequent FISH detection process, so that the stability of the FISH detection of the cell filter membrane sample is ensured.
In some of these embodiments, the lysing red blood cells essentially comprises the steps of: (1) Mixing the collected human peripheral blood sample with the erythrocyte lysate to perform erythrocyte lysis treatment; (2) centrifuging to discard supernatant and reserving cell sediment; (3) Sequentially adding PBS and a fixing agent, mixing by vortex, and standing at room temperature to obtain a cell suspension sample from which red blood cells are removed.
The human peripheral blood rare cell EGFR gene amplification detection method mainly comprises the following steps:
obtaining a cell filter membrane sample by adopting any rare cell enrichment method,
EGFR gene amplification detection is carried out on the filter membrane;
The EGFR gene amplification detection on the filter membrane mainly comprises the following steps: (1) Baking the cell filter membrane sample, soaking and dehydrating the cell filter membrane sample by absolute ethyl alcohol, and airing the cell filter membrane sample at room temperature; (2) Pretreating a cell filter membrane sample by using any one of the pretreatment liquids; (3) digesting the cell filter sample with a digestive enzyme; (4) Hybridization of the cell filter samples using FISH probes against EGFR genes and control genes; (5) post-hybridization washing; (6) DAPI counterstain; (7) fluorescent microscope observation.
The FISH probe of the control gene is a FISH probe of chromosome 7.
In some of these embodiments, in the hybridization reaction: denaturation at 85 ℃ for 8+/-0.5 min, hybridization at 42 ℃ for 3-18 hours; preferably, the hybridization is performed for 8.+ -. 0.5 minutes at 85.+ -. 1 ℃ and 4.+ -. 0.1 hours at 42.+ -. 1 ℃.
In some of these embodiments, the pretreatment conditions are: incubating for 3-10 min at room temperature.
In some of these embodiments, preferably, the pretreatment solution comprises 50mM NaOH solution and 0.5% (v/v) Triton X-100, and the pretreatment conditions are: incubate 5.+ -. 0.5 min at room temperature.
The inventor of the present invention has found that the pretreatment of the cell filter membrane sample by the pretreatment liquid can loosen the whole cell structure of the cells in a short time at room temperature, so that the nucleic acid combined with the protein is also exposed and released, and the DNA in the cells can be effectively hybridized with the fluorescent probe during the subsequent FISH detection.
Compared with the prior art, the invention has the following beneficial effects:
the inventor of the invention develops a human peripheral blood rare cell EGFR gene amplification detection method, which comprises a simple hydrophilic treatment step, and the filter membrane for cell filtration can have good hydrophilicity and biocompatibility by using a hydrophilic modifier specially developed for the detection method of the invention to carry out hydrophilic treatment on the filter membrane for cell filtration, and the cell suspension sample after red blood cells are removed can be used for effectively filtering white blood cell enriched rare cells by using the filter membrane for filter membrane filtration, so that cells trapped on the filter membrane are paved on the filter membrane as much as possible, a large number of cells are stacked on the filter membrane, and the problems of poor imaging effect, high fluorescence background and the like in subsequent FISH detection caused by cell stacking are avoided; meanwhile, the hydrophilic treatment step can ensure that rare cells trapped on the filter membrane are not easy to fall off from the membrane in the subsequent FISH detection process, thereby ensuring the stability of the FISH detection of the cell filter membrane sample.
The detection method of the invention also comprises a simple pretreatment step, and the pretreatment liquid specially developed for the detection method of the invention is used for pretreating the cell filter membrane sample, so that the cell membrane can be destroyed to a certain extent, the basic form and structure of the cell nucleus can be maintained, the cell surface tension can be increased at room temperature to promote the whole cell structure of the cell to be loosened in a short time, the DNA and protein in the cell can be denatured, the nucleic acid combined with the protein can be exposed and released, and the DNA in the cell can be effectively hybridized with the fluorescent probe during the subsequent FISH detection.
The inventor of the invention also developed a kit for the detection method of the invention and a use method of the kit, compared with conventional cell FISH, the kit has good balance in hybridization effect and effective cell maintenance and can maintain higher stability; meanwhile, the method is simple and time-saving in operation, the whole detection process can be completed within 9 hours, the fluorescent background is low, the hybridization effect is good, and therefore noninvasive real-time accurate monitoring of EGFR gene amplification state can be well achieved.
Drawings
FIG. 1 is a schematic diagram showing the negative and positive detection results of EGFR gene amplification of the present invention.
FIG. 2 is a graph showing comparison of the results of the hydrophilic treatment with the hydrophilic modifier and the non-hydrophilic treatment of the filter membrane of example 5;
FIG. 3 is a graph showing the comparison of the detection results of the pretreatment of the cell filter samples in example 8 with the pretreatment liquid of the present invention and the conventional pretreatment for digestion.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Experimental methods, in which specific conditions are not noted in the examples below, are generally carried out according to conventional conditions, for example, green and Sambrook-s.A.fourth edition, molecular cloning, A.laboratory Manual (Molecular Cloning: A Laboratory Manual), published in 2013, or according to the conditions recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The present invention will be described in further detail with reference to specific examples.
Example 1 method for detecting EGFR Gene amplification in rare cells of human peripheral blood
The method for detecting the EGFR gene amplification of the rare cells in the peripheral blood of the human body mainly comprises the following four procedures:
1. collecting a human peripheral blood sample
The collection of the human peripheral blood sample is to collect blood in a human vein by using a vacuum blood collection tube, and the blood is usually 5 ml/tube.
2. Lysing erythrocytes
The method for lysing erythrocytes mainly comprises the following steps: (1) Mixing the collected human peripheral blood sample with the erythrocyte lysate to perform erythrocyte lysis treatment; (2) centrifuging to discard supernatant and reserving cell sediment; (3) Sequentially adding PBS and a fixing agent, mixing by vortex, and standing at room temperature to obtain a cell suspension sample from which red blood cells are removed.
3. Filtration membrane filtration
The filter membrane filtration mainly comprises the following steps: (1) Performing hydrophilic treatment on a filter membrane in the filter by using a hydrophilic modifier; (2) Transferring the cell suspension sample from which the red blood cells are removed to a filter for filtering with a filter membrane to remove the white blood cells and enrich rare cells; (3) adding 4% formaldehyde solution, fixing at room temperature, and removing liquid; (4) And adding PBS, washing and soaking for three times, removing liquid, and taking out the filter membrane from the filter to obtain a cell filter membrane sample.
The hydrophilic modifier in the filter membrane filtering step (1) consists of a solvent and a solute, wherein the solvent is water or buffer solution such as sodium acetate and sodium citrate, the solute comprises 0.03-10% (w/v) of carboxyethyl cellulose, 0.05-5% (w/v) of betaine, 0.05-1% (v/v) of surfactant, 0.02-0.2% (v/v) of polylysine and 0.01-0.1% (v/v) of preservative, the surfactant is selected from any one of Triton X-100, tween20, tween80 and S10, the preservative is ProClin 300, and the pH value of the hydrophilic modifier is 6.0-8.0.
In this example, the hydrophilic modifier comprises 10mM sodium citrate buffer (pH 6.0), 0.5% (w/v) carboxyethylcellulose, 0.1% (w/v) betaine, 0.5% (v/v) Tween80, 0.1% (v/v) polylysine, and 0.05% (v/v) ProClin 300, at a pH of 6.8;
the hydrophilic treatment in the filter membrane filtration step (1) specifically comprises the following steps: adding 200-600 mu l of hydrophilic modifier into the filter membrane of the filter, infiltrating the filter membrane for 3-10 minutes at room temperature, filtering to remove all liquid, and drying at 50 ℃; preferably, the hydrophilic treatment in this embodiment specifically includes: 400 μl of hydrophilic modifier was added to the filter membrane, the membrane was immersed for 5 minutes at room temperature, all the liquid was removed by filtration, and the mixture was dried at 50deg.C.
4. EGFR gene amplification assay on Filter
The EGFR gene amplification detection on the filter membrane mainly comprises the following steps: (1) Baking the cell filter membrane sample, soaking and dehydrating the cell filter membrane sample by absolute ethyl alcohol, and airing the cell filter membrane sample at room temperature; (2) Pretreating a cell filter membrane sample by using a pretreatment liquid; (3) digesting the cell filter sample with a digestive enzyme; (4) Hybridization of the cell filter samples using FISH probes for EGFR gene and chromosome 7; (5) post-hybridization washing; (6) DAPI counterstain; (7) fluorescent microscope observation. Wherein,
The pretreatment liquid for EGFR gene amplification detection step (2) on the filter membrane comprises 25-100 mM alkaline solution and 0.3-1% (v/v) surfactant, wherein the alkaline solution is 25-100 mM NaOH or KOH solution, and the surfactant is selected from any one of Triton X-100, tween20, tween80 and NP 40.
In this example, the pretreatment solution included 50mM NaOH solution and 0.5% (v/v) Triton X-100.
The pretreatment conditions of EGFR gene amplification detection step (2) are carried out on a filter membrane: incubating for 3-10 min at room temperature. In this embodiment, the pretreatment conditions are: incubate for 5 minutes at room temperature.
Unless otherwise indicated, other reagents involved in the detection method described in this example, such as red blood cell lysate, PBS, fixative, 4% formaldehyde solution, digestive enzymes, FISH probes for EGFR gene and chromosome 7, washing solution, DAPI, etc., are all general reagents in the art and can be prepared or purchased by themselves. The filter for filtering the filter membrane and the filter membrane related to the detection method in this embodiment are also general materials in the field, wherein the filter structure and the composition can refer to the filter device related to the chinese patent application CN103468568A, the filter membrane is the same as the filter membrane related to the chinese patent application CN104178454A, CN112111456a, the filter membrane is selected from a polycarbonate filter membrane, a fiber filter membrane, a nylon filter membrane or a plastic film, and the pore size of the filter membrane is 5-8 μm. In this example, the filter was a polycarbonate filter with a filter pore size of 8. Mu.m.
Example 2 preparation and use of the kit for the detection method of example 1
The present example provides a kit for the method for detecting EGFR gene amplification of human peripheral blood rare cells described in example 1, which mainly comprises eight components of red blood cell lysate, fixative, filter, hydrophilic modifier, pretreatment liquid, digestive enzyme, EGFR/CEN7 probe solution and counterstain liquid, and the formulation/composition and preservation conditions of the eight components are shown in Table 1.
Table 1 formulation/composition of kit Components and storage conditions
The kit of this example may further comprise seven other components including PBS, 4% formaldehyde solution, absolute ethanol, 70% ethanol, 85% ethanol, washing solution I and washing solution II, and their formulations/compositions and storage conditions are shown in Table 2.
Table 2 formulation/composition of other components of kit and storage conditions
The preparation method of the kit in the embodiment is as follows: the components of the kit are prepared according to the formulations/compositions of the above tables 1 and 2, the components of the kit are respectively packaged and assembled according to the specification of the kit, and the kit is preserved according to the preservation condition requirements of the components of the kit.
The embodiment also provides a use method of the kit, and specific steps are shown in Table 3.
TABLE 3 specific steps of the method of Using the kit of this example
The kit and the use method thereof are used for detecting peripheral blood samples (with the numbers of 1-10 and from lung cancer patients) of 10 tumor patients, and simultaneously, a commercially available EGFR gene amplified positive lung cancer cell strain NCI-H1975 and EGFR gene amplified negative healthy human leucocytes are respectively used as a positive control and a negative control. Respectively taking 1000 NCI-H1975 cells and healthy human leucocytes (determined by a cell counter), uniformly mixing, uniformly dividing the samples into 5 parts (numbers 11-15 and 16-20), detecting, reading 50 cells with DAPI blue fluorescent signals in each cell strain sample, counting the number of cells expressing red/green fluorescence, and simultaneously counting the number of EGFR gene amplification positive and negative cells and the average number of fluorescence points of the red/green fluorescence, wherein the number of cells in the sample is selected by automatic scanning of a fluorescence microscope. The specific results are shown in Table 4.
TABLE 4 sample test results
The detection finds that the clinical detection result of the tumor patient sample is consistent with the detection result of the kit used for the detection method of the invention; aiming at different cell samples to be detected, the detection results are the same each time, and the detection results show that the EGFR amplification detection method for the human peripheral blood rare cells has good specificity and sensitivity, and can realize clinical sample detection. The kit for the EGFR amplification detection method of the peripheral blood rare cells has 100% of coincidence rate with clinical detection results, which shows that the detection system and the detection method of the kit can accurately detect the EGFR gene amplification condition in the peripheral blood rare cells of patients and have high accuracy.
EXAMPLE 3 determination of the concentration of the hydrophilic modifier Components
1. Design of concentration of hydrophilic modifier constituent components
Aiming at the EGFR gene amplification detection method of human peripheral blood rare cells, in order to improve the hydrophilicity of a filter membrane, the trapped cells are not easy to fall off from the filter membrane, and a hydrophilic treatment step of a hydrophilic modifier is introduced into the filter flow of the filter membrane. The hydrophilic modifier consists of a solvent and a solute, wherein the solvent is water or buffer solution such as sodium acetate and sodium citrate, the solute comprises carboxyethyl cellulose, betaine, a surfactant, polylysine and a preservative ProClin 300, and the surfactant is selected from any one of Triton X-100, tween20, tween80 and S10.
To determine the concentrations of the hydrophilic modifier components, the solvent was exemplified by 10mM sodium citrate buffer (pH 6.0), the surfactant was exemplified by Tween80, and experimental groups 1-5 were designed, and the detection results were compared with the kit described in example 2 except that the concentrations of the hydrophilic modifier components were different for each experimental group, as shown in Table 5.
TABLE 5 design of hydrophilic modifier compositions
2. Sample detection
The experiment was performed using commercially available cell line NCI-H1975 and healthy human leukocytes. NCI-H1975 cells and healthy human leukocyte samples were prepared 15 times each, numbered 1-15 and 16-30 in sequence, 100 cells each (determined by a cell counter). The hydrophilic modifier prepared by the design, the filter membrane with proper pore size and the kit components described in the example 2 are adopted, samples 1 to 30 are detected according to the using method of the kit described in the example 2, each sample of each cell strain in each experimental group is detected 3 times, all cells with DAPI blue fluorescence signals in 100 cells of each sample are read, the number of cells expressing red/green fluorescence is counted, and the number of cells positive and negative to EGFR gene amplification and the average fluorescence point number of the red/green fluorescence are counted, wherein the number of cells in the sample is selected by automatic scanning of a fluorescence microscope. The specific results are shown in Table 6.
TABLE 6 comparison of the results of detection of hydrophilic modifiers of different compositional concentrations
From the detection results, the experimental groups 2, 3 and 4 can realize accurate detection, and all EGFR amplified positive cells and EGFR amplified negative cells can be detected correctly, wherein the fluorescent signals detected by the experimental group 3 are more stable, and the detection effect is better; while both experimental group 1 and experimental group 5 had a certain degree of cell omission and the EGFR-amplified positive cells were detected as EGFR-amplified negative cells (see samples 2 and 15 of Table 6), it was presumed that the reason was probably that the hydrophilic treatment of the filter with the hydrophilic modifier at the concentration of the constituent components could not completely ensure that the trapped cells were not detached from the filter, or could not completely avoid the false negative results caused by the cell stacking.
The experimental design for determining the concentration of the components of the hydrophilic modifier by taking water or sodium acetate buffer solution as a solvent and/or Triton X-100, tween20 and S10 as surfactants is similar to the experimental design, the experimental result is similar to the experimental result, and specific design and experimental data are omitted. The experimental design for determining the pH value of the hydrophilic modifier is similar to the experimental design, the specific design and experimental data are omitted, and the result shows that the hydrophilic modifier can be accurately detected when the pH value is 6.0-8.0, and the detected fluorescent signal is more stable and the detection effect is better when the pH value is 6.8.
In view of the above experimental results, the hydrophilic modifier in the detection method of the invention uses water or buffer solutions such as sodium acetate and sodium citrate as solvents, and the solute comprises 0.03% -10% (w/v) of carboxyethylcellulose, 0.05% -5% (w/v) of betaine, 0.05% -1% (v/v) of surfactant, 0.02% -0.2% (v/v) of polylysine, and 0.01% -0.1% (v/v) of preservative ProClin 300, and the pH value is 6.0-8.0, wherein the surfactant is any one of Triton X-100, tween20, tween80 and S10; preferably, the hydrophilic modifier in the detection method of the present invention comprises 10mM sodium citrate buffer (pH 6.0), 0.5% (w/v) carboxyethylcellulose, 0.1% (w/v) betaine, 0.5% (v/v) Tween80, 0.1% (v/v) polylysine, and 0.05% (v/v) ProClin 300, at pH 6.8.
Example 4 determination of hydrophilic treatment method
1. Design of hydrophilic treatment method
In order to determine the hydrophilic treatment method of the present invention in the EGFR gene amplification test method for human peripheral blood rare cells, taking hydrophilic treatment of 400. Mu.l of a hydrophilic modifier as an example, experimental groups 1 to 5 were designed, and each experimental group was identical except for the hydrophilic treatment operation, and the optimal conditions were determined in examples 1 to 2. The test results were compared and the specific designs are shown in Table 7.
TABLE 7 design of hydrophilic treatment method
2. Sample detection
The experiment was performed using commercially available cell line NCI-H1975 and healthy human leukocytes. NCI-H1975 cells and healthy human leukocyte samples were prepared 15 times each, numbered 1-15 and 16-30 in sequence, 100 cells each (determined by a cell counter). The samples 1 to 30 were examined by using a filter membrane with a suitable pore size and the kit components described in example 2, according to the above-mentioned hydrophilic treatment method and the kit and using method described in example 2, 3 samples of each cell line of each experimental group were examined, all cells having DAPI blue fluorescent signal in 100 cells of each sample were read, the number of cells expressing red/green fluorescence was counted, and the numbers of EGFR gene amplification positive and negative cells and the average number of fluorescence points of red/green fluorescence thereof were counted, wherein the number of cells in the samples was selected by fluorescent microscope auto-scanning. The specific results are shown in Table 8.
TABLE 8 comparison of test results for different hydrophilic treatments
From the detection results, the experimental groups 2, 3 and 4 can realize accurate detection, and all EGFR amplified positive cells and EGFR amplified negative cells can be detected correctly, wherein the fluorescent signals detected by the experimental group 3 are more stable, and the detection effect is better; while both experimental group 1 and experimental group 5 had a certain degree of cell omission, and experimental group 5 had a phenomenon that EGFR-amplified positive cells were detected as EGFR-amplified negative cells (see sample 13 of table 8), presumably because the hydrophilic treatment method may not completely ensure that the trapped cells were not detached from the filter membrane, or may not completely avoid false negative results caused by cell stacking.
The experimental design for the hydrophilic treatment method for 200. Mu.l and 600. Mu.l of the hydrophilic modifier was determined similarly to the above experimental design, and the experimental results were also similar to the above experimental results, and specific design and experimental data were omitted.
In view of the above experimental results, the hydrophilic treatment in the detection method of the present invention is specifically as follows: adding 200-600 mu l of hydrophilic modifier into the filter membrane of the filter, infiltrating the filter membrane for 3-10 minutes at room temperature, filtering to remove all liquid, and drying at 50 ℃; preferably, the hydrophilic treatment in the detection method of the present invention specifically comprises: 400 μl of hydrophilic modifier was added to the filter membrane, the membrane was immersed for 5 minutes at room temperature, all the liquid was removed by filtration, and the mixture was dried at 50deg.C.
EXAMPLE 5 verification of hydrophilic treatment Effect of hydrophilic modifier of the present invention
1. Design for verifying hydrophilic treatment effect of hydrophilic modifier
In order to verify the hydrophilic treatment effect of the hydrophilic modifier in the EGFR gene amplification detection method for the peripheral blood rare cells of the human body, an experimental group 1 and an experimental group 2 are designed, the filter membrane of the experimental group 1 is subjected to hydrophilic treatment by using the hydrophilic modifier, the filter membrane of the experimental group 2 is not subjected to hydrophilic treatment, and the detection effect is compared, and the specific design is shown in a table 9. The other detection methods and the condition design of the kit are all the optimal conditions determined in examples 1 to 2.
TABLE 9 design for hydrophilic modifier hydrophilic treatment effect verification
Experimental group Design for verifying hydrophilic treatment effect
Group 1 Hydrophilic treatment of filter membranes with the hydrophilic modifier of the invention
Group 2 The filter membrane is not subjected to hydrophilic treatment
2. Sample detection
In this example, experiments were performed using peripheral blood samples from 5 patients with known EGFR-amplified positive tumors (numbers 1-5) and 5 patients with known EGFR-amplified negative tumors (numbers 6-10). The kit components described in example 2 are adopted, samples 1 to 10 are detected according to the design and the using method of the kit described in example 2, cells with DAPI blue fluorescence signals in each sample are read, the number of cells expressing red/green fluorescence is counted, and the number of positive and negative cells amplified by EGFR genes and the average fluorescence point number of the red/green fluorescence are counted, wherein the number of cells in the samples is selected by automatic scanning of a fluorescence microscope. In addition, the leukocyte removal rate was counted based on the number of leukocytes removed by filtration and the number of remaining leukocytes. The specific results are shown in Table 10 and FIG. 2.
Table 10 hydrophilic modifier hydrophilic treatment effect verification test results
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From the above detection results, the accurate detection can be realized in experiment group 1, all EGFR amplification positive patients detect EGFR amplification positive cells, all EGFR amplification negative patients detect EGFR amplification negative cells only, the number of EGFR amplification positive/negative cells detected by the EGFR amplification positive patients is more, the number of fluorescence signal points is more, the leukocyte filtration rate is 99.7% -99.8%, and the fluorescence background is low (see experiment group 1 in fig. 2), while the accurate detection can be realized in experiment group 2, but compared with experiment group 1, the number of EGFR amplification positive/negative cells detected by the EGFR amplification positive patients is relatively less, the number of fluorescence signal points detected by the EGFR amplification positive/negative cells is relatively less, the leukocyte filtration rate is 99.2% -99.6%, and the fluorescence background is relatively high (see experiment group 2 in fig. 2), and it is presumed that the possible that experiment group 2 is not subjected to hydrophilic treatment steps of a hydrophilic modifier, cells on a filter membrane are easier to stack, so that the number of detected cells is less than that in experiment group 1, and the fluorescence background is lower than that the experiment group 1 is filtered by experiment group 1. The hydrophilic modifier in the detection method has good hydrophilic treatment effect, can ensure that the filter membrane has good hydrophilicity and biocompatibility, can effectively filter out rare cells enriched in white blood cells, and avoids a large number of stacking of cells on the filter membrane, thereby avoiding the problems of poor imaging effect, high fluorescent background and the like in the subsequent FISH detection caused by cell stacking; meanwhile, rare cells on the filter membrane are not easy to fall off in the subsequent FISH detection process, so that the stability of the FISH detection of the cell filter membrane sample is ensured.
EXAMPLE 6 determination of the concentration of the Components of the pretreatment liquid
1. Design of concentration of components of pretreatment liquid
Aiming at the EGFR gene amplification detection method of human peripheral blood rare cells, the pretreatment step of the pretreatment liquid is introduced in the EGFR gene amplification detection flow carried out on a filter membrane so as to replace the conventional digestion pretreatment step. The pretreatment liquid comprises alkaline solution NaOH or KOH and a surfactant, wherein the surfactant is selected from any one of Triton X-100, tween20, tween80 and NP 40.
In order to determine the concentration of the components of the pretreatment liquid, the alkaline solution is exemplified by NaOH solution, the surfactant is exemplified by Triton X-100, experimental groups 1-5 are designed, the components of each experimental group are the same as those of the kit described in example 2 except that the concentration of the components of the pretreatment liquid is different, and the detection effect is compared, and the specific design is shown in Table 11.
TABLE 11 design of pretreatment liquid composition
Experimental group The components of the pretreatment liquid
Group 1 10mM NaOH solution, 0.1% (v/v) Triton X-100
Group 2 25mM NaOH solution, 0.3% (v/v) Triton X-100
Group 3 50mM NaOH solution, 0.5% (v/v) Triton X-100
Group 4 100mM NaOH solution, 1% (v/v) Triton X-100
Group 5 125mM NaOH solution, 1.2% (v/v) Triton X-100
2. Sample detection
The experiment was performed using commercially available cell line NCI-H1975 and healthy human leukocytes. 3000 NCI-H1975 cells and healthy human leucocytes (determined by a cell counter) are taken respectively, and after uniform mixing, the samples are divided into 15 parts respectively, and the numbers are 1-15 and 16-30 in sequence. The pretreatment solution with proper pore size, the pretreatment solution prepared by the design and the kit components described in the embodiment 2 are adopted, samples 1 to 30 are detected according to the using method of the kit described in the embodiment 2, each sample of each cell strain in each experimental group is detected 3 times, 50 cells with DAPI blue fluorescence signals in each sample are read, the number of cells expressing red/green fluorescence is counted, and simultaneously, the number of cells positive and negative to EGFR gene amplification and the average fluorescence point number of the red/green fluorescence are counted, wherein the number of cells in the sample is selected by automatic scanning of a fluorescence microscope. The specific results are shown in Table 12.
TABLE 12 comparison of the detection results of pretreatment solutions of different composition concentrations
From the detection results, the experimental groups 2, 3 and 4 can realize accurate detection, and all EGFR amplified positive cells and EGFR amplified negative cells can be detected correctly, wherein the fluorescent signals detected by the experimental group 3 are more stable, and the detection effect is better; whereas, both the experimental group 1 and the experimental group 5 have the phenomena of missing detection of EGFR amplification positive and/or negative cells with different degrees, and have the phenomenon that EGFR amplification positive cells are detected as EGFR amplification negative cells (see samples 3 and 13 of table 12), the number of the fluorescence signal points detected in the EGFR amplification positive cells is smaller than that of the experimental groups 2, 3 and 4, presumably, the reason is that the concentration of the components of the pretreatment liquid is too low or too high, the pretreatment effect is poor, the hybridization effect of DNA in the cells and the fluorescence probes is poor, the detected fluorescence signals are weakened, and even false negative results and/or missing detection of negative and positive cells are caused.
The experimental design for determining the concentration of the components of the alkaline solution KOH and/or the pretreatment solution taking Tween20, tween80 and NP40 as the surfactant is similar to the experimental design, the experimental result is similar to the experimental result, and the specific design and experimental data are omitted. The results showed that accurate detection was achieved when the pretreatment liquid composition was 25 to 100mM alkaline solution and 0.3 to 1% (v/v) surfactant, wherein the detection effect was better when the pretreatment liquid composition was 50mM alkaline solution and 0.5% (v/v) surfactant, and the detection effect was better when the pretreatment liquid composition was 50mM NaOH solution and 0.5% (v/v) Triton X-100 than when the pretreatment liquid composition was other alkaline solution and/or other surfactant at the same concentration.
In view of the experimental results, the pretreatment liquid in the detection method comprises 25-100 mM alkaline solution and 0.3-1% (v/v) surfactant, wherein the alkaline solution is 25-100 mM NaOH or KOH solution, and the surfactant is selected from any one of Triton X-100, tween20, tween80 and NP 40; preferably, the pretreatment liquid in the detection method of the present invention comprises 50mM NaOH solution and 0.5% (v/v) Triton X-100.
EXAMPLE 7 determination of pretreatment Condition
1. Design of pretreatment conditions
In order to determine pretreatment conditions in the detection method for EGFR gene amplification of human peripheral blood rare cells, experimental groups 1-6 are designed, all experimental groups are identical except that the pretreatment conditions are different, and the condition designs of other detection methods and kits are the optimal conditions determined in examples 1-2.
The test results were compared and the specific designs are shown in Table 13.
TABLE 13 design of pretreatment conditions
Experimental group Pretreatment conditions Experimental group Pretreatment conditions
Group 1 Incubation at room temperature for 1 min Group 2 Incubation at room temperature for 3 min
Group 3 Incubation at room temperature for 5 min Group 4 Incubation at room temperature for 10 min
Group 5 Incubation at room temperature for 15 min Group 6 Incubation at 42℃for 5 min
2. Sample detection
The experiment was performed using commercially available cell line NCI-H1975 and healthy human leukocytes. 3600 NCI-H1975 cells and healthy human leucocytes (determined by a cell counter) were taken respectively, and after mixing, the samples were divided equally into 18 parts, numbered 1 to 18 and 19 to 36 in sequence. The detection of samples 1 to 36 was carried out by using a filter membrane with a suitable pore size and the kit components described in example 2, according to the pretreatment conditions described above and the method for using the kit described in example 2, 3 samples of each cell line of each experimental group were examined, 50 cells with DAPI blue fluorescence signal in each sample were read, the number of cells expressing red/green fluorescence was counted, and the number of EGFR gene amplification positive and negative cells and the average number of fluorescence points of red/green fluorescence thereof were counted, wherein the number of cells in the samples was selected by automatic scanning with a fluorescence microscope. The specific results are shown in Table 14.
TABLE 14 comparison of test results for different pretreatment conditions
From the detection results, the experimental groups 2, 3 and 4 can realize accurate detection, and all EGFR amplified positive cells and EGFR amplified negative cells can be detected correctly, wherein the fluorescent signals detected by the experimental group 3 are more stable, and the detection effect is better; while each of the experimental groups 1, 5 and 6 had different degrees of EGFR-amplified positive and/or negative cell omission phenomenon, and there were the phenomena that EGFR-amplified positive cells were detected as EGFR-amplified negative cells (see samples 1, 14 and 17 of Table 14), the number of fluorescence signal points detected in EGFR-amplified positive cells was smaller than that in the experimental groups 2, 3 and 4, presumably due to improper pretreatment time or improper temperature, the pretreatment effect was poor, the hybridization effect of DNA in cells hybridized with the fluorescence probes was poor, the detected fluorescence signals were weakened, and even false negative results and/or the omission of negative and positive cells was caused. Thus, the pretreatment conditions in the detection method of the present invention are: incubating for 3-10 minutes at room temperature; preferably, the pretreatment conditions in the detection method of the present invention are: incubate for 5 minutes at room temperature.
EXAMPLE 8 pretreatment Effect verification of the pretreatment liquid of the invention
1. Design for verifying pretreatment effect of pretreatment liquid
In order to verify the pretreatment effect of the pretreatment liquid in the EGFR gene amplification detection method for the peripheral blood rare cells of the human body, an experimental group 1 and an experimental group 2 are designed, the pretreatment liquid is used for pretreatment of the cell filter membrane sample of the experimental group 1, the conventional pretreatment for digestion is carried out on the cell filter membrane sample of the experimental group 2, and the detection effect is compared, and the specific design is shown in a table 15.
TABLE 15 design for pretreatment effect verification of pretreatment liquid
2. Sample detection
The experiment was performed using commercially available cell line NCI-H1975 and healthy human leukocytes. 2000 NCI-H1975 cells and healthy human leucocytes (determined by a cell counter) were taken respectively, and after mixing, the samples were divided equally into 10 parts, numbered 1 to 10 and 11 to 20 in sequence. The method of the above design and the method of the use of the kit of the example 2 are adopted to detect samples 1 to 20, 5 samples of each cell line of each experimental group are detected, 50 cells with DAPI blue fluorescence signals in each sample are read, the number of cells expressing red/green fluorescence is counted, and the number of positive and negative cells amplified by EGFR genes and the average fluorescence point number of the red/green fluorescence are counted, wherein the number of cells in the samples is selected by automatic scanning of a fluorescence microscope. The specific results are shown in Table 16 and FIG. 3.
Table 16 pretreatment effect verification test results of pretreatment liquid
From the above detection results, both experiment group 1 and experiment group 2 can realize accurate detection, and all EGFR amplification positive and negative cells can be detected correctly, but compared with experiment group 2, the number of fluorescence signal points detected by EGFR amplification positive cells in experiment group 1 is more, the fluorescence signal is more stable, and the detection effect is better (see table 16 and fig. 3). This demonstrates that the pretreatment of the pretreatment solution of the present invention provides a better pretreatment than conventional pretreatment prior to digestion, which allows the whole cell structure of the cells to be loosened at room temperature in a short period of time, allowing the nucleic acid bound to the protein to be exposed and released, facilitating efficient hybridization of the DNA in the cells with fluorescent probes during the subsequent FISH detection.
Example 9 Effect of hybridization reaction procedure on the detection Effect of the kit
1. Design of hybridization reaction program
In order to examine the influence of the hybridization reaction procedure on the detection effect of the kit for use in the detection method of the present invention, experimental groups 1 to 5 were designed, each of which was identical except for the hybridization reaction procedure, and the detection effect was compared, and specific designs are shown in Table 17.
TABLE 17 design of hybridization reaction program
Experimental group Hybridization reaction procedure
Group 1 Denaturation at 85℃for 8 min, hybridization at 42℃for 2 h
Group 2 Denaturation at 85℃for 8 min, hybridization at 42℃for 3 h
Group 3 Denaturation at 85℃for 8 min, hybridization at 42℃for 4 h
Group 4 Denaturation at 85℃for 8 min, hybridization at 42℃for 18 h
Group 5 Denaturation at 85℃for 8 min, hybridization at 42℃for 24 h
2. Sample detection
The experiment was performed using commercially available cell line NCI-H1975 and healthy human leukocytes. 3000 NCI-H1975 cells and healthy human leucocytes (determined by a cell counter) are taken respectively, and after uniform mixing, the samples are divided into 15 parts respectively, and the numbers are 1-15 and 16-30 in sequence. The detection of samples 1 to 30 is carried out according to the method of using the kit described in example 2 and the hybridization procedure described above by using a filter membrane with a suitable pore size and the kit components described in example 2, 3 samples of each cell line of each experimental group are examined, 50 cells with DAPI blue fluorescence signals in each sample are read, the number of cells expressing red/green fluorescence is counted, and simultaneously the number of EGFR gene amplification positive and negative cells and the average number of fluorescence points of red/green fluorescence thereof are counted, wherein the number of cells in the sample is selected by automatic scanning by a fluorescence microscope. The specific results are shown in Table 18.
TABLE 18 comparison of the detection results of different hybridization reaction procedures
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From the detection results, the experimental groups 2, 3 and 4 can realize accurate detection, and all EGFR amplification positive cells and EGFR amplification negative cells can be detected correctly, wherein the fluorescent signals detected by the experimental group 3 are more stable, and the detection effect is better; whereas experimental group 1 has the phenomenon of missed detection of EGFR amplification positive or negative cells, the number of the detected fluorescent signal points in EGFR amplification positive cells is smaller than that in experimental groups 2, 3 and 4, and the phenomenon that EGFR amplification positive cells are detected as EGFR amplification negative cells (see sample 3 of table 18) is presumed to be caused by too short hybridization time, so that DNA in the cells cannot be completely hybridized with fluorescent probes, the detected fluorescent signal is weakened, and even false negative results and/or the phenomenon of missed detection of negative and positive cells are caused; although the experimental group 5 can correctly detect all EGFR amplified positive cells, and the number of the detected fluorescence signal points is more than that of the experimental groups 2, 3 and 4, the phenomenon that EGFR amplified negative cells are detected as EGFR amplified positive cells (see sample 29 of Table 18) is presumed to be caused by overlong hybridization time, so that nonspecific fluorescence signals are generated, the detected fluorescence signals are increased, and even false positive results are caused. Thus, the kit for use in the detection method of the present invention is used in a method wherein the hybridization reaction procedure is denaturation at 85℃for 8 minutes, hybridization at 42℃for 3 to 18 hours, preferably denaturation at 85℃for 8 minutes and hybridization at 42℃for 4 hours.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (8)

1. The hydrophilic modifier is characterized by comprising a solvent and a solute, wherein the solvent is sodium citrate buffer solution, and the solute comprises 0.03-10% (w/v) of carboxyethyl cellulose, 0.05-5% (w/v) of betaine, 0.05-1% (v/v) of surfactant, 0.02-0.2% (v/v) of polylysine and has a pH value of 6.0-8.0; the surfactant is Tween80; the hydrophilic modifier also comprises 0.01% -0.1% (v/v) preservative, wherein the preservative is ProClin 300.
2. The hydrophilic modifier of claim 1, wherein the solute comprises 0.1% to 5 (w/v) carboxyethylcellulose, 0.05% to 3% (w/v) betaine, 0.1% to 0.8% (v/v) surfactant, 0.05% to 0.15% (v/v) polylysine, and the pH is 6.0 to 7.5.
3. The hydrophilic modifier of claim 1, wherein the solvent of the hydrophilic modifier is 10mM sodium citrate buffer at ph6.0, and the solute comprises: 0.2 to 0.8 percent (w/v) of carboxyethyl cellulose, 0.08 to 0.15 percent (w/v) of betaine, 0.2 to 0.7 percent (v/v) of surfactant, 0.08 to 0.12 percent (v/v) of polylysine and the pH value is between 6.7 and 6.9.
4. The kit for detecting the EGFR gene amplification of the human peripheral blood rare cells is characterized by mainly comprising the following components: a hydrophilic modifier according to any one of claims 1 to 3, and a pretreatment liquid comprising 25 to 100mM alkaline solution and 0.3% to 1% (v/v) surfactant; the alkaline solution is 45-55 mM NaOH solution or KOH solution; the surfactant is Triton X-100.
5. The kit for detecting EGFR gene amplification of human peripheral blood rare cells according to claim 4, wherein the pretreatment solution comprises 45-55 mM alkaline solution and 0.3% -0.6% (v/v) surfactant.
6. The rare cell enrichment method for the EGFR gene amplification detection of the human peripheral blood rare cells is characterized by comprising the following steps:
S1, obtaining a human peripheral blood sample, and lysing erythrocytes to obtain a cell suspension sample from which erythrocytes are removed;
s2, filtering by a filter membrane; obtaining a pretreated cell filter membrane sample;
the step S2, the filter membrane filtration mainly comprises the following steps: (1) Hydrophilizing a filter membrane in a filter with the hydrophilic modifier as claimed in any one of claims 1 to 4; (2) Transferring the cell suspension sample after removing the red blood cells into a filter for filtering with a filter membrane to remove the rare cells enriched in white blood cells; (3) adding formaldehyde solution, fixing at room temperature, and removing liquid; (4) And adding PBS, washing and soaking for three times, removing liquid, and taking out the filter membrane from the filter to obtain a cell filter membrane sample.
7. The rare cell enrichment method for human peripheral blood rare cell EGFR gene amplification assay according to claim 6, wherein the hydrophilic treatment comprises the steps of: 200-600 mul of the hydrophilic modifier is added into the filter membrane of the filter, the filter membrane is soaked for 3-10 minutes at room temperature, all liquid is removed by filtration, and the filter membrane is dried at 50+/-1 ℃.
8. The rare cell enrichment method for human peripheral blood rare cell EGFR gene amplification assay according to claim 7, wherein the hydrophilic treatment is as follows: adding 350-450 mu l of hydrophilic modifier into the filter membrane of the filter, soaking the filter membrane for 5+/-0.5 minutes at room temperature, filtering to remove all liquid, and drying at 50+/-1 ℃.
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