CN111484976A - Lung cancer circulating tumor cell detection kit and detection system - Google Patents
Lung cancer circulating tumor cell detection kit and detection system Download PDFInfo
- Publication number
- CN111484976A CN111484976A CN201910080739.0A CN201910080739A CN111484976A CN 111484976 A CN111484976 A CN 111484976A CN 201910080739 A CN201910080739 A CN 201910080739A CN 111484976 A CN111484976 A CN 111484976A
- Authority
- CN
- China
- Prior art keywords
- lung cancer
- solution
- circulating tumor
- egfr
- cell detection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Oncology (AREA)
- Pathology (AREA)
- Biophysics (AREA)
- Hospice & Palliative Care (AREA)
- Cell Biology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a lung cancer circulating tumor cell detection system, which is used for detecting circulating tumor cells which are separated from a lung cancer solid tumor and enter peripheral blood, and is characterized by comprising the following components: the lung cancer circulating tumor cell detection kit comprises an EGFR (epidermal growth factor receptor) immunomagnetic ball, a connector group and a primer group, wherein the EGFR immunomagnetic ball is prepared by firstly mixing an emulsifier GHDC and skeleton material cholesterol of a link molecule with a magnetic bead solution, then adding a matrix material DOPC solution and a surfactant HQCMC solution for room temperature ultrasound, and finally adding an EGFR antibody. Therefore, the detection system can be used for carrying out accurate, rapid and high-flux gene sequence analysis on the CTC which is very rare in blood, thereby providing essential information of tumor formation, differentiation and metastasis and providing more information for tumor diagnosis, prognosis judgment, metastasis and individualized treatment.
Description
Technical Field
The invention relates to the fields of biotechnology and biomedicine, in particular to a lung cancer circulating tumor cell detection kit and a lung cancer circulating tumor cell detection system.
Background
The lung cancer is one of the highest malignant tumors in the global morbidity and mortality, the annual morbidity of the lung cancer in China is estimated to reach 100 ten thousand people in 2025 years, wherein 85-90 percent of the lung cancer mainly comprises non-small cell lung cancer (NSC L C), and about 60-70 percent of lung cancer patients have metastasis during initial diagnosis due to low early detection rate of the lung cancer and no specific and detailed clinical representation, so the chance of early surgical treatment is lost.
Circulating Tumor Cells (CTC) refer to Tumor cells released from a solid Tumor focus (primary focus, metastatic focus) into peripheral blood circulation due to spontaneous or diagnosis and treatment operations, and are important reasons for postoperative recurrence and distant metastasis of malignant Tumor patients. In recent years, the role of CTC in the process of malignant tumor metastasis is increasingly concerned, the application of CTC in clinical diagnosis and treatment is more and more extensive with the continuous maturation of technical means, the detection of CTC can be effectively applied to the in vitro early diagnosis of tumors, and the CTC has important clinical significance and wide application prospect.
High-throughput sequencing (NGS), also known as massively parallel sequencing, can sequence millions or even billions of DNA molecules simultaneously, achieves the goal of large-scale, high-throughput sequencing, and is a revolutionary advance following Sanger sequencing. At present, in clinical tumor practice, NGS is mainly applied to driving gene sequencing and is an important link for accurate diagnosis and treatment of tumors. Different from Sanger sequencing technology, the NGS technology achieves higher sensitivity and accuracy by repeatedly sequencing DNA fragments in the same region, has high flux and high automation degree, can complete sequencing of hundreds of billions of basic groups in a short time, and makes it possible to complete human whole genome sequencing within a few days. The NGS technology plays an important role in promoting the development of molecular biology due to high accuracy, high speed and low cost, provides a new means for the research of tumor molecular biology, and especially plays a more important role in the field of tumor molecular diagnosis.
There are many methods for detecting CTCs, but conventional methods cannot provide the essence of tumor formation, differentiation and metastasis due to the very rare amount and low purity of CTCs in circulating peripheral blood.
Disclosure of Invention
The present invention has been made to solve the above problems, and an object of the present invention is to provide a lung cancer circulating tumor cell detection kit and a lung cancer circulating tumor cell detection system.
The invention provides a lung cancer circulating tumor cell detection kit, which is used for detecting circulating tumor cells which are separated from a lung cancer solid tumor and enter peripheral blood, and has the characteristics that: EGFR immune magnetic ball for capturing circulating tumor cells is prepared by the following steps: step one, according to dimethyl octadecyl epoxypropyl ammonium chloride: mixing dimethyl octadecyl epoxypropyl ammonium chloride and cholesterol in a mass ratio of 1:1 to obtain a first mixture; step two, according to the magnetic bead solution after ethanol removal: CH (CH)2Cl21: 1-1: 5 volume ratio, dissolving the magnetic bead solution after ethanol removal in CH2Cl2Obtaining a first solution; step three, according to the first solution: first mixture ═ 1: 5-1: 10, mixing the first solution and the first mixture to obtain a second solution; step four, according to the dioleoyl phosphatidylcholine solution: carboxymethyl chitosan hexadecyl quaternary ammonium salt solution: second solution ═ 1: 1: 5-1: 1: 12, mixing the dioleoylphosphatidylcholine solution, the carboxymethyl chitosan hexadecyl quaternary ammonium salt solution and the second solution to obtain a third solution; step five, performing room temperature ultrasonic treatment on the third solution, and removing residual CH2Cl2Obtaining a fourth solution; step six, according to the EGFR antibody: fourth solution ═ 1: 16-1: and (3) mixing the EGFR antibody and the fourth solution according to the mass-to-volume ratio of 20 to obtain the EGFR immunomagnetic ball.
The kit for detecting circulating tumor cells of lung cancer provided by the invention can also compriseSuch features are: the preparation process of the carboxymethyl chitosan hexadecyl quaternary ammonium salt in the carboxymethyl chitosan hexadecyl quaternary ammonium salt solution is as follows: according to epoxy hexadecimal quaternary ammonium salt: carboxymethyl chitosan is dissolved in dd H together with epoxy hexadecane quaternary ammonium salt and carboxymethyl chitosan in a mass ratio of 30:12And adding isopropanol with the same volume into the mixture O, stirring the mixture at 50-55 ℃ overnight, dialyzing the mixture for 24 hours by using a dialysis bag with the molecular weight of 10000 Dalton, and freeze-drying the dialyzed mixture to obtain the carboxymethyl chitosan hexadecyl quaternary ammonium salt.
The lung cancer circulating tumor cell detection kit provided by the invention also has the following characteristics: the staining agent is used for staining circulating tumor cells captured by the EGFR immunomagnetic spheres and comprises a first staining agent, a second staining agent and a third staining agent, wherein the first staining agent is 4', 6-diamidino-2-phenylindole, the second staining agent is fluorescein isothiocyanate labeled by cytokeratin 8, 18 and 19, and the third staining agent is phycoerythrin labeled by leukocyte common antigen CD 45.
The kit for detecting circulating tumor cells of lung cancer provided by the present invention may further include: an adapter group and a primer group for constructing a high-throughput sequencing library, wherein the adapter group comprises a first adapter and a second adapter, and the sequence of the first adapter is
5'-gatcggaagagcacacgtctgaactccagtcactgcagtatctcgtatgccgtcttctgcttg-3', the sequence of the second linker is
5'-aatgatacggcgaccaccgagatctacactctttccctacacgacgctcttccgatct-3', the primer group comprises an upstream primer and a downstream primer, the sequence of the upstream primer is
5'-CAAGCAGAAGACGGCATACGA-3', the sequence of the downstream primer is
5’-CAAGCAGAAGACGGCATACGA-3’。
The lung cancer circulating tumor cell detection kit provided by the invention also has the following characteristics: wherein the size of the high-throughput sequencing library is 300-450 bp.
The kit for detecting circulating tumor cells of lung cancer provided by the invention can also compriseCharacterized in that, it also includes: RNase-Free ddH2O and 2 × Blood Direct PCR Master Mix.
The invention provides a lung cancer circulating tumor cell detection system, which is used for detecting circulating tumor cells which are separated from a lung cancer solid tumor and enter peripheral blood, and is characterized by comprising the following components: the kit comprises a lung cancer circulating tumor cell detection kit and a high-throughput sequencer, wherein the lung cancer circulating tumor cell detection kit is any one of the lung cancer circulating tumor cell detection kits.
Action and Effect of the invention
According to the lung cancer circulating tumor cell detection kit provided by the invention, the EGFR immune magnetic ball is prepared by firstly mixing emulsifier dimethyl octadecyl epoxypropyl ammonium chloride and skeleton material cholesterol of linked molecules with a magnetic bead solution, then adding a matrix material dioleoyl phosphatidylcholine solution and a surfactant carboxymethyl chitosan hexadecyl quaternary ammonium salt solution for room temperature ultrasound, and finally adding an EGFR antibody, so that the EGFR immune magnetic ball can carry out specific targeted rapid separation on Circulating Tumor Cells (CTC) in blood to capture the CTC, is convenient for subsequent detection and analysis of the captured CTC, and particularly carries out high-throughput sequencing analysis to provide more information for tumor diagnosis, prognosis judgment, metastasis and individualized treatment.
In addition, in the preparation process of the EGFR immune magnetic bead, the EGFR antibody is added finally, so that the problem that the subsequent steps after the antibody is added in advance have adverse effects on the activity of the antibody so that the activity of the antibody is reduced and the capture efficiency of the prepared EGFR immune magnetic bead on the lung cancer circulating tumor cells is influenced is solved, therefore, the EGFR immune magnetic bead has high activity and high purity, can capture CTCs in peripheral blood of a detected person to the maximum extent with strong specificity and high sensitivity, and reduces the capture leakage of the CTCs as much as possible. In addition, the whole preparation process of the EGFR immune magnetic ball can be completed at room temperature, the preparation process is simple, and the production cost is low.
According to the lung cancer circulating tumor cell detection system, the lung cancer circulating tumor cell detection kit and the high-throughput sequencer are included, CTCs in blood can be captured through the lung cancer circulating tumor cell detection kit, then a high-throughput sequencing library is constructed for the captured CTCs, and then high-throughput sequencing is carried out through the high-throughput sequencer, so that accurate, rapid and high-throughput gene sequence analysis can be carried out on the CTCs which are very rare in the blood, thereby providing essential information of tumor formation, differentiation and metastasis, and providing more information for tumor diagnosis, prognosis judgment, metastasis and individualized treatment.
Drawings
FIG. 1 is a graph showing the results of a simulation experiment in example two of the present invention;
FIG. 2 is a flowchart of lung cancer circulating tumor cell detection in the third embodiment of the present invention;
FIG. 3 is a graph showing the results of immunofluorescence identification of CTC in example three of the present invention;
FIG. 4 is a statistical chart of CTC immunofluorescence counts in example three of the present invention; and
FIG. 5 is a flowchart of lung cancer circulating tumor cell detection in four embodiments of the present invention.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the efficacy of the invention easy to understand, the following embodiments are specifically described with reference to the drawings.
< example one >
The embodiment provides a lung cancer circulating tumor cell detection system, which is used for capturing Circulating Tumor Cells (CTCs) which are separated from a lung cancer solid tumor and enter peripheral blood, and identifying and detecting genes of the captured CTCs, and the system comprises the following steps: a lung cancer circulating tumor cell detection kit and a high-throughput sequencer.
The lung cancer circulating tumor cell detection kit in the embodiment comprises an EGFR (epidermal growth factor receptor) immunomagnetic ball, a staining agent, a joint group, a primer group and RNase-Free ddH2O and 2 × Blood Direct PCR Master Mix.
The EGFR immune magnetic ball of the embodiment is used for capturing the circulating tumor cells, and the preparation process is as follows:
step one, weighing 10mg of each of dimethyloctadecyl oxypropylammonium chloride (GHDC) and cholesterol, mixing the GHDC and the cholesterol to obtain a first mixture, and adding the first mixture into a ground pear-shaped bottle.
Step two, measuring 1.0m L magnetic bead solution after ethanol removal to dissolve in 2.0m L CH2Cl2To obtain a first solution.
And step three, pouring the first solution into a ground pear-shaped bottle to mix the first solution and the first mixture to obtain a second solution. At this time, the second solution: mixture 3: 20 volume to mass ratio.
And step four, weighing 200 mu L10 mg/m L Dioleoylphosphatidylcholine (DOPC) solution and 200 mu L10 mg/m L carboxymethyl chitosan hexadecyl quaternary ammonium salt (HQCMC) solution into the second solution by using a pipette, and uniformly mixing to obtain a third solution, wherein the volume ratio of the DOPC solution to the HQCMC solution to the second solution is 1: 1: 7.5.
The preparation process of the HQCMC solution comprises the following steps: according to epoxy hexadecimal quaternary ammonium salt: carboxymethyl chitosan ═ 30:1, the epoxy hexadecyl quaternary ammonium salt and the carboxymethyl chitosan are dissolved in dd H together2In O, simultaneously with dd H2Stirring O isopropanol with the same volume at 50-55 ℃ overnight, dialyzing for 24H by using a dialysis bag with the molecular weight of 10000 Dalton, and replacing dd H once every two hours in the dialysis process2And O, freeze-drying after dialysis is finished to obtain the HQCMC. The HQCMC solution is an aqueous solution of HQCMC.
Putting the pear-shaped bottle containing the third solution into an ultrasonic cell crushing instrument, starting the instrument, wherein ultrasonic parameters comprise power of 27%, ultrasonic frequency of 2s, interval of 1s, total time of 6min, temperature of 25 ℃, pausing and rapidly adding 6m L dd H after ultrasonic frequency is 1min2And O, continuing ultrasound till the end, and removing residual CH by rotary evaporation after the ultrasound is finished2Cl2Obtaining a fourth solution;
and a sixth step of mixing an Epidermal Growth Factor Receptor (EGFR) antibody and the fourth solution at a mass-to-volume ratio of 50 μ g: 0.8m L-50 μ g: 1m L, and vortexing the mixture for 8 hours to mix the mixture sufficiently to obtain EGFR immunomagnetic spheres, wherein in the present example, the EGFR and the fourth solution are at a mass-to-volume ratio of 50 μ g: 0.8m L.
The stain of this example stains CTCs captured by EGFR immunomagnetic spheres during CTC identification and enumeration, the stain comprising a first stain, a second stain, and a third stain. The first stain is 4', 6-diamidino-2-phenylindole (DAPI), the second stain is fluorescein isothiocyanate (CK8-FITC, CK18-FITC and CK19-FITC) labeled by cytokeratin 8, 18 and 19, and the third stain is phycoerythrin (CD45-PE) labeled by leukocyte common antigen CD 45.
The linker group is used for constructing a high-throughput sequencing library and comprises a first linker and a second linker. Wherein the sequence of the first linker is
5'-gatcggaagagcacacgtctgaactccagtcactgcagtatctcgtatgccgtcttctgcttg-3', the sequence of the second linker is
5'-aatgatacggcgaccaccgagatctacactctttccctacacgacgctcttccgatct-3' are provided. In this embodiment, the first and second connectors are mixed together in equal volumes.
The primer group is used for constructing a high-throughput sequencing library and comprises an upstream primer and a downstream primer. Wherein, the sequence of the upstream primer is 5'-CAAGCAGAAGACGGCATACGA-3', and the sequence of the downstream primer is 5'-CAAGCAGAAGACGGCATACGA-3'.
The high-throughput sequencer in this embodiment is an instrument for high-throughput sequencing of a high-throughput sequencing library, and may be any one of the high-throughput sequencers in the prior art.
< example two >
This example is a capture efficiency simulation experiment performed on the lung cancer circulating tumor cell detection kit of the first example by combining human lung cancer cells (H1975).
The specific process is as follows:
in step S1-1, human lung cancer cells (H1975) were cultured in a cell culture solution for a certain period of time, a certain amount of the cells were added to an EP tube and PBS was added, 10 μ L diluted solution was applied to a counting plate, and the number of H1975 cells was read out and the cell concentration was calculated (cell concentration ═ N × 1000 dilution).
In step S1-2, the counted H1975 cells are grouped (5 groups), different numbers of H1975 cells are added into each group to form a gradient of 10, 50, 100, 250 and 500H 1975 cell numbers, respectively, and a certain amount of PBS is added into each group to make up to 7.5m L.
Step S1-3, 20. mu. L EGFR immunomagnetic beads were added to each of the 5 groups of H1975 cells, and incubated at room temperature for 30min, and mixed uniformly every 5 min.
And step S1-4, after the incubation is finished, inserting the EP tube into a magnetic separation frame for adsorption for 15min, then discarding the supernatant, adding a first coloring agent, a second coloring agent and a third coloring agent which are respectively 20 mu L, uniformly mixing and dyeing for 15min in a dark place.
And step S1-5, after dyeing is completed, adding 1m L PBS for washing and magnetic separation for 10min, then removing supernatant, adding 1m L double distilled water for washing and magnetic separation for 10min, then removing supernatant, adding 20 mu L deionized water into an EP tube for resuspending the cell-magnetic sphere mixed solution, uniformly coating the mixture on an anti-shedding glass slide, and observing and counting under a fluorescence microscope after liquid drops are automatically dried.
FIG. 1 is a graph showing the results of a simulation experiment in example two of the present invention.
As shown in FIG. 1, in this example, the capture rate of human lung cancer cells (H1975) by EGFR immunomagnetic sphere pair in 5 groups of experimental groups was as high as 80% or more.
< example three >
The embodiment provides a method for detecting circulating tumor cells of lung cancer, namely, the kit for detecting circulating tumor cells of lung cancer in the first embodiment is used for separating CTC from blood of a patient with lung cancer, and the separated CTC is subjected to an identification and counting experiment.
In the embodiment, the blood sample collection is that the blood of cancer patients in hospitals is collected by a medical anticoagulant blood collection tube, the anticoagulant is EDTA ■ K and is stored at 2.4 ℃, the blood is prevented from being frozen in the processes of storage, treatment and transportation, and the detection is carried out within 48h (a sample collection unit has scientific research cooperation projects, is approved by the ethical committee of hospitals, and signs an informed consent with the patients).
FIG. 2 is a flowchart of lung cancer circulating tumor cell detection in the third embodiment of the present invention.
As shown in fig. 2, the specific process of the method for detecting circulating tumor cells of lung cancer in this embodiment is as follows:
and step S2-1, preparing a peripheral blood sample, namely centrifuging 3.25m L whole blood of a lung cancer tumor patient collected in an anticoagulation blood collection tube for 10min at 2500rpm, putting the supernatant in a 15m L EP tube, adding PBS (with the pH value of 7.4) with the same volume as the supernatant, and fully and uniformly mixing to obtain the peripheral blood sample.
And S2-2, separating the CTC from the EGFR immunomagnetic spheres, namely adding the EGFR immunomagnetic spheres 20 mu L into a peripheral blood sample, incubating at room temperature for 30min, uniformly mixing every 5min, inserting an EP tube into a magnetic separation frame to adsorb for 15min, sucking and removing supernatant, taking out the EP tube to obtain the EGFR immunomagnetic spheres adsorbing the CTC, which are called CTC-EGFR magnetic spheres for short, and then adding 1m L PBS to carry out magnetic separation and washing on the CTC-EGFR magnetic spheres for 1 time.
And step S2-3, performing CTC immunostaining, namely, adding a first staining agent, a second staining agent and a third staining agent into the washed CTC-EGFR magnetic ball, wherein each staining agent is 20 mu L, and uniformly mixing and staining for 15min in a dark place.
And S2-4, performing CTC immunofluorescence identification and counting, namely adding 1m L PBS into the dyed mixed solution, washing and magnetically separating for 10min, then discarding the supernatant, adding 1m L double distilled water, washing and magnetically separating for 10min, then discarding the supernatant, adding 20 mu L deionized water into an EP tube, re-suspending the cell-magnetic sphere mixed solution, uniformly coating the mixture on an anti-falling glass slide, automatically airing the liquid drops, and observing and counting under a fluorescence microscope, wherein the result is shown in figure 2.
FIG. 3 is a graph showing the results of immunofluorescence identification of CTC in example three of the present invention.
As shown in FIG. 3, the DAPI test was positive (cells were blue), the CK19-FITC test was positive (cells were green), and the CD45-PE test was negative (field of view was dark, cells could not be observed) under the irradiation of a fluorescence microscope, thus indicating that the cells were lung cancer cells. Then, 3 lung cancer cells as shown in FIG. 2 were detected in a fluorescence microscope.
In addition, 30 whole blood samples of the same lung cancer tumor patients were tested according to the above method of this example, and the results are shown in FIG. 4.
FIG. 4 is a statistical chart of CTC immunofluorescence counting results in example three of the present invention.
As shown in FIG. 4, it can be seen that the number of lung cancer cells in the peripheral blood of the patient with lung cancer tumor is about three in this test system, indicating that the detection method is reliable and stable.
< example four >
The embodiment provides a method for detecting circulating tumor cells of lung cancer, which is to perform CTC separation on blood of a lung cancer patient by using the system for detecting circulating tumor cells of lung cancer in the embodiment one, and perform a high-throughput sequencing experiment on the separated CTCs.
Blood sample collection in this example: the medical anticoagulant blood collection tube is used for collecting blood of cancer patients in hospitals, and the anticoagulant is EDTA-K2. Storing at 4 deg.C, avoiding freezing during storage, processing and transportation, and detecting within 48h (sample collection unit has scientific research cooperative project, and has approved by hospital ethics committee, and has signed informed consent with patient).
FIG. 5 is a flowchart of lung cancer circulating tumor cell detection in the fourth embodiment of the present invention.
As shown in fig. 5, the specific process of the method for detecting circulating tumor cells of lung cancer in this embodiment is as follows:
and step S3-1, preparing a peripheral blood sample, namely centrifuging 3.25m L whole blood of a lung cancer tumor patient collected in an anticoagulation blood collection tube for 10min at 2500rpm, putting the supernatant in a 15m L EP tube, adding PBS (with the pH value of 7.4) with the same volume as the supernatant, and fully and uniformly mixing to obtain the peripheral blood sample.
And S3-2, separating the CTC from the EGFR immunomagnetic spheres, namely adding the EGFR immunomagnetic spheres 20 mu L into a peripheral blood sample, incubating at room temperature for 30min, uniformly mixing every 5min, inserting an EP tube into a magnetic separation frame to adsorb for 15min, sucking and removing supernatant, taking out the EP tube to obtain the EGFR immunomagnetic spheres adsorbing the CTC, which are called CTC-EGFR magnetic spheres for short, and then adding 1m L PBS to carry out magnetic separation and washing on the CTC-EGFR magnetic spheres for 1 time.
Step S3-3, obtaining genome DNA. Specifically, the DNA in CTC was extracted by treating the CTC-EGFR magnetic spheres with the Genomic DNA Purification Kit, Cat.No.: B0007, EZBioscience Kit, and the procedure of the EZBioscience Kit was followed, and the obtained DNA was stored in a centrifuge tube and refrigerated.
And step S3-4, constructing a high-throughput sequencing library. That is to say that the first and second electrodes,
and (1) crushing the DNA obtained in the step S3-3 to about 300bp by using covaris S220, and recovering the fragmented product by using a Qiagen PCR kit, specifically according to the operation instruction of the Qiagen kit.
Smashing instrument parameter setting:
and recovering the broken product DNA and purifying the product.
Step (2), fragmenting the product DNA 30. mu. L, 10. mu. Blunting Buffer 5. mu. L, 1mM dNTP 10. mu. L kit Enzyme Mix 1. mu. L, ddH2O9. mu. L, Total volume 50. mu. L, incubation at 30 ℃ for 30 min. the filling-in product was purified with 1.6 volumes of Ampure XP beads (DNA purification kit Zymo Genomic DNAClean)&Concentrator cargo number: d4011) In that respect
Adding dATP into the purified product obtained in the step (2), and taking the filling-in product 20 mu L, 100mM dATP 1 mu L, 10. about. NEB Buffer23 mu L, Klenow exo (NEB) (50000units/m L) 0.5 mu L, ddH2O5.5 μ L, total volume 30 μ L, incubation at 37 deg.C for 30min, and heat inactivation at 75 deg.C for 20 min.
And (4) adding a dATP product and adding a joint.
The specific conditions were 24. mu. L, 2. mu. Quick L alignment Buffer 30. mu. L, Quick T4DNA L igase 1.2. mu. L, 8. mu.M linker set 5. mu. L, total volume 60. mu. L, ligation for 30min at room temperature.
And (5) quantifying the purified ligation products by using the Qubit, and mixing the ligation products of different samples in an equal amount according to the sequencing data amount.
Step (6), preparing 1% agarose gel electrophoresis, cutting the mixed ligation products to select fragment size, and adopting a gel recovery kit of Qiagen company for recovery; and carrying out Qubit quantification on the recovered product for the next PCR amplification.
And (3) PCR amplification:
the reaction system and conditions are as follows, DNA sample 3 mu L, upstream primer 1 mu L, downstream primer 1 mu L, 2 × PhusionPCR Master Mix 25 mu L, H2O20 μ L, 50 μ L in total, provided that 95 ℃ for 1min, then 12 cycles of 94 ℃ for 10s, 60 ℃ for 30s, and 72 ℃ for 10s, and finally an extension of 5min at 72 ℃.
And (3) purifying the PCR product by using equal volume AWP ure XP Beads twice, wherein the purification process is strictly carried out according to the use instruction, and taking out 1 mu L for 2100 detection, wherein the result shows that the size of the obtained high-throughput sequencing library is generally 200-350 bp.
Step S3-5, sequencing using a high throughput sequencer.
< example five >
The embodiment provides a method for detecting circulating tumor cells of lung cancer, which is to use the system for detecting circulating tumor cells of lung cancer in the embodiment one to separate CTCs from blood of a patient with lung cancer, and to perform identification, counting and high-throughput sequencing experiments on the separated CTCs.
Blood sample collection in this example: the medical anticoagulant blood collection tube is used for collecting blood of cancer patients in hospitals, and the anticoagulant is EDTA-K2. Storing at 4 deg.C, avoiding freezing during storage, processing and transportation, and detecting within 48h (sample collection unit has scientific research cooperative project, and has approved by hospital ethics committee, and has signed informed consent with patient).
The specific process of the lung cancer circulating tumor cell detection method in the embodiment is as follows:
and step S4-1, preparing a peripheral blood sample, namely centrifuging the 7m L whole blood of a lung cancer tumor patient collected in an anticoagulation blood collection tube at 2500rpm for 10min, putting the supernatant in a 15m L EP tube, adding PBS (with the pH value of 7.4) with the same volume as the supernatant, and fully and uniformly mixing the mixture to obtain the peripheral blood sample.
At step S4-2, the peripheral blood sample was divided equally into two aliquots, one for CTC isolation and identification according to the procedure in example three, and the other for CTC isolation and high throughput sequencing according to the procedure in example four.
Effects and effects of the embodiments
According to the lung cancer circulating tumor cell detection kit provided by the embodiment, the EGFR immunomagnetic sphere is prepared by firstly mixing an emulsifier dimethyl octadecyl epoxypropyl ammonium chloride and a skeleton material cholesterol of a linking molecule with a magnetic bead solution, then adding a matrix material dioleoyl phosphatidylcholine solution and a surfactant carboxymethyl chitosan hexadecyl quaternary ammonium salt solution for room temperature ultrasound, and finally adding an EGFR antibody, so that the EGFR immunomagnetic sphere can perform specific targeted rapid separation on Circulating Tumor Cells (CTCs) in blood to capture the CTCs, and further facilitates subsequent detection and analysis of the captured CTCs, especially high-throughput sequencing analysis, and provides more information for tumor diagnosis, prognosis judgment, metastasis and individualized treatment.
In addition, in the preparation process of the EGFR immunomagnetic beads, the EGFR antibody is added last, so that the problem that the subsequent steps after the antibody is added in advance adversely affect the activity of the antibody to reduce the activity of the antibody, thereby affecting the capture efficiency of the prepared EGFR immunomagnetic beads on the lung cancer circulating tumor cells is avoided, and therefore, in a simulation system, the capture efficiency of the EGFR immunomagnetic beads on the lung cancer circulating tumor cells in the embodiment can be as high as more than 80%. In addition, the whole preparation process of the EGFR immune magnetic ball can be completed at room temperature, the preparation process is simple, and the production cost is low.
Further, since the carboxymethyl chitosan hexadecyl quaternary ammonium salt (carboxymethyl chitosan hexadecyl quaternary ammonium salt) in the first embodiment is prepared by the preparation method in the present embodiment, the carboxymethyl chitosan hexadecyl quaternary ammonium salt (carboxymethyl chitosan hexadecyl quaternary ammonium salt) in the present embodiment has high purity and high activity, and is the best choice for preparing high-quality and high-efficiency EGFR immunomagnetic beads.
Further, the kit for detecting circulating tumor cells of lung cancer in the first embodiment further comprises a first stain, a second stain and a third stain, wherein the first stain is DAPI, the second stain comprises CK8-FITC, CK18-FITC and CK19-FITC, and the third stain is CD45-PE, so that captured CTCs can be stained by the three stains, the staining effect is good, the staining effect is not faded for a long time, and the captured CTCs can be identified and counted by a fluorescence microscope. In addition, the second stain comprises fluorescein isothiocyanate markers of three cytokeratins, namely CK8-FITC, CK18-FITC and CK19-FITC, so that specific staining of CTC can be ensured to the greatest extent, and identification and counting results of the CTC are more accurate.
According to the lung cancer circulating tumor cell detection system related to the embodiment, because the lung cancer circulating tumor cell detection kit and the high-throughput sequencer are included, CTCs in blood can be captured by the lung cancer circulating tumor cell detection kit, then a high-throughput sequencing library is constructed for the captured CTCs, and then high-throughput sequencing is performed by the high-throughput sequencer, so that accurate, rapid and high-throughput gene sequence analysis can be performed on the very rare CTCs in the blood, thereby providing essential information of tumor formation, differentiation and metastasis, and providing more information for tumor diagnosis, prognosis judgment, metastasis and individualized treatment.
In conclusion, the system for detecting circulating tumor cells of lung cancer according to the present embodiment can rapidly and accurately detect the circulating tumor cells in the blood of the subject from the cellular level and the genetic level.
The above embodiments are preferred examples of the present invention, and are not intended to limit the scope of the present invention.
Claims (7)
1. A lung cancer circulating tumor cell detection kit for detecting circulating tumor cells that have escaped from a solid lung cancer tumor and entered into peripheral blood, comprising:
EGFR immune magnetic ball for capturing the circulating tumor cells, which is prepared by the following process:
step one, according to dimethyl octadecyl epoxypropyl ammonium chloride: mixing dimethyl octadecyl epoxypropyl ammonium chloride and cholesterol in a mass ratio of 1:1 to obtain a first mixture;
step two, according to the magnetic bead solution after ethanol removal: CH (CH)2Cl21: 1-1: 5 volume ratio, dissolving the magnetic bead solution after ethanol removal in CH2Cl2Obtaining a first solution;
step three, according to the first solution: the first mixture is 1: 5-1: 10, mixing the first solution and the first mixture to obtain a second solution;
step four, according to the dioleoyl phosphatidylcholine solution: carboxymethyl chitosan hexadecyl quaternary ammonium salt solution: the second solution is 1: 1: 5-1: 1: 12, mixing the dioleoylphosphatidylcholine solution, the carboxymethyl chitosan hexadecyl quaternary ammonium salt solution and the second solution to obtain a third solution;
step five, removing residual CH after carrying out room temperature ultrasonic treatment on the third solution2Cl2Obtaining a fourth solution;
step six, according to the EGFR antibody: the fourth solution is 1: 16-1: and 20, mixing the EGFR antibody and the fourth solution to obtain the EGFR immunomagnetic spheres.
2. The lung cancer circulating tumor cell detection kit according to claim 1, characterized in that:
wherein the preparation process of the carboxymethyl chitosan hexadecyl quaternary ammonium salt in the carboxymethyl chitosan hexadecyl quaternary ammonium salt solution is as follows:
according to epoxy hexadecimal quaternary ammonium salt: carboxymethyl chitosan is dissolved in dd H together with epoxy hexadecane quaternary ammonium salt and carboxymethyl chitosan in a mass ratio of 30:12And adding isopropanol with the same volume into the mixture O, stirring the mixture at 50-55 ℃ overnight, dialyzing the mixture for 24 hours by using a dialysis bag with the molecular weight of 10000 Dalton, and freeze-drying the dialyzed mixture to obtain the carboxymethyl chitosan hexadecyl quaternary ammonium salt.
3. The circulating tumor cell detection kit for lung cancer according to claim 1, further comprising:
a staining agent for staining the circulating tumor cells captured by the EGFR immunomagnetic sphere, comprising a first staining agent, a second staining agent, and a third staining agent,
wherein the first coloring agent is 4', 6-diamidino-2-phenylindole,
the second staining agent is fluorescein isothiocyanate marked by cytokeratin 8, 18 and 19,
the third stain is phycoerythrin marked by leukocyte common antigen CD 45.
4. The kit for detecting circulating tumor cells of lung cancer according to claim 1, further comprising:
an adaptor group and a primer group for constructing a high-throughput sequencing library,
wherein the joint group comprises a first joint and a second joint,
the sequence of the first linker is 5'-gatcggaagagcacacgtctgaactccagtcactgcagtatctcgtatgccgtcttctgcttg-3',
the second connector has the sequence 5'-aatgatacggcgaccaccgagatctacactctttccctacacgacgctcttccgatct-3',
the primer group comprises an upstream primer and a downstream primer,
the sequence of the upstream primer is 5'-CAAGCAGAAGACGGCATACGA-3',
the sequence of the downstream primer is 5'-CAAGCAGAAGACGGCATACGA-3'.
5. The kit for detecting circulating tumor cells of lung cancer according to claim 4, wherein:
wherein the size of the high-throughput sequencing library is 300-450 bp.
6. The circulating tumor cell detection kit for lung cancer according to claim 1, further comprising:
RNase-Free ddH2o and 2 × Blood Direct PCR Master Mix.
7. A system for detecting circulating tumor cells of lung cancer, which is used for detecting circulating tumor cells that have escaped from a solid tumor of lung cancer and enter peripheral blood, comprising:
a lung cancer circulating tumor cell detection kit and a high-throughput sequencer,
the lung cancer circulating tumor cell detection kit is the lung cancer circulating tumor cell detection kit according to any one of claims 1 to 6.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910080739.0A CN111484976A (en) | 2019-01-28 | 2019-01-28 | Lung cancer circulating tumor cell detection kit and detection system |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910080739.0A CN111484976A (en) | 2019-01-28 | 2019-01-28 | Lung cancer circulating tumor cell detection kit and detection system |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111484976A true CN111484976A (en) | 2020-08-04 |
Family
ID=71791165
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910080739.0A Pending CN111484976A (en) | 2019-01-28 | 2019-01-28 | Lung cancer circulating tumor cell detection kit and detection system |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111484976A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113789302A (en) * | 2021-09-15 | 2021-12-14 | 南昌大学第二附属医院 | Enrichment and separation kit for lung cancer circulating tumor cells |
| CN114276992A (en) * | 2021-12-14 | 2022-04-05 | 南通举健生物科技有限公司 | Complete exosome separation and purification kit and detection analysis method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102234658A (en) * | 2010-04-23 | 2011-11-09 | 上海市肿瘤研究所 | Target quaternary ammonium salt cationic polymer lipid gene carrier, preparation method and application thereof |
| CN105785005A (en) * | 2016-04-13 | 2016-07-20 | 杭州华得森生物技术有限公司 | Circulating tumor cell detection kit and application thereof |
| CN106635995A (en) * | 2017-03-10 | 2017-05-10 | 亚能生物技术（深圳）有限公司 | Circulating tumor cell negative enrichment method |
| CN108031442A (en) * | 2017-12-29 | 2018-05-15 | 猎源（上海）生物医药科技有限公司 | A kind of nano-lipid magnetic ball and preparation method thereof, the kit of separation and Extraction dissociative DNA and its application |
-
2019
- 2019-01-28 CN CN201910080739.0A patent/CN111484976A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102234658A (en) * | 2010-04-23 | 2011-11-09 | 上海市肿瘤研究所 | Target quaternary ammonium salt cationic polymer lipid gene carrier, preparation method and application thereof |
| CN105785005A (en) * | 2016-04-13 | 2016-07-20 | 杭州华得森生物技术有限公司 | Circulating tumor cell detection kit and application thereof |
| CN106635995A (en) * | 2017-03-10 | 2017-05-10 | 亚能生物技术（深圳）有限公司 | Circulating tumor cell negative enrichment method |
| CN108031442A (en) * | 2017-12-29 | 2018-05-15 | 猎源（上海）生物医药科技有限公司 | A kind of nano-lipid magnetic ball and preparation method thereof, the kit of separation and Extraction dissociative DNA and its application |
Non-Patent Citations (4)
| Title |
|---|
| 朱立平，陈学清: "《免疫学常用实验方法》", 31 March 2000 * |
| 梁晓飞,王汉杰,田惠,罗浩,常津: "新型载药壳聚糖季铵盐的合成、结构与性能", 《物理化学学报》 * |
| 石远凯等: "《临床路径释义 上 肿瘤疾病分册 2018版》", 31 August 2018, 中国协和医科大学出版社 * |
| 闫国超等: "《肿瘤疾病临床检验与诊断》", 30 April 2017, 科学技术文献出版社 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113789302A (en) * | 2021-09-15 | 2021-12-14 | 南昌大学第二附属医院 | Enrichment and separation kit for lung cancer circulating tumor cells |
| CN114276992A (en) * | 2021-12-14 | 2022-04-05 | 南通举健生物科技有限公司 | Complete exosome separation and purification kit and detection analysis method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106650312B (en) | Device for detecting copy number variation of circulating tumor DNA | |
| CN110191962A (en) | The sequencing and analysis of allochthon associated nucleic acid | |
| CN107849607A (en) | The single-molecule sequencing of plasma dna | |
| CN103572378A (en) | Establishment method for small fragmental DNA (Deoxyribose Nucleic Acid) library based on Ion ProtonTM sequencing platform and application of establishment method | |
| CN106845154B (en) | A device for FFPE sample copy number variation detects | |
| CN111487404B (en) | Body fluid tumor cell DNA extraction kit | |
| CN114317762B (en) | Three-marker composition for detecting early liver cancer and kit thereof | |
| CN109576347A (en) | The sequence measuring joints of the label containing unimolecule and the construction method of sequencing library | |
| CN111172263A (en) | Reference substance applied to noninvasive prenatal detection and preparation method thereof | |
| CN111484976A (en) | Lung cancer circulating tumor cell detection kit and detection system | |
| CN113293196A (en) | Single cell nucleus extraction method suitable for frozen tissue | |
| CN115927565A (en) | Establishment and application of method for detecting pathogenic microorganisms in CAR-T cell product based on mNGS | |
| WO2022161294A1 (en) | Construction method and use of medium-throughput single-cell copy number library | |
| CN106350609B (en) | A kind of reagent, detection method and application for vesicular stomatitis virus detection | |
| US20180355408A1 (en) | Selective protection of nucleic acids | |
| CN109266653B (en) | Reagent, device and method for capturing drug-resistant heterogeneous circulating tumor cells and performing gene analysis | |
| CN113528531B (en) | Aptamer for detecting human lung cancer cell strain A549 extracellular vesicles and application thereof | |
| CN115125238A (en) | Method for separating and purifying tumor extracellular vesicle DNA | |
| CN113249441B (en) | Reference substance for detecting pathogenic microorganisms infected with blood influenza and preparation method thereof | |
| CN114891856A (en) | Large-queue sample single cell enrichment and mixed sample library construction sequencing method | |
| CN113528507A (en) | Kit for extracting chicken blood genome DNA by high-throughput rapid paramagnetic particle method and extraction method | |
| CN114657184A (en) | Multivalent aptamer functionalized DNA nano-structure probe and preparation method and application thereof | |
| CN112391444A (en) | Application of mesenchymal stem cells with different functional characteristics in treating different diseases | |
| CN112063615A (en) | Genome DNA extracting solution, genome DNA extracting method and application thereof | |
| WO2020128538A1 (en) | Novel method for identification of viruses and diagnostic kit using the same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |