A brain glioma detection kit based on circulating tumor cells

Abstract

The present invention relates to the technical field of tumor detection, and specifically to a glioma detection kit based on circulating tumor cells. It is used to detect circulating tumor cells of glioma; it includes a transfection reagent and a surface antigen detection reagent; the transfection reagent includes a construct, the construct includes a human telomerase reverse transcriptase promoter for regulating the construct to generate a detection signal; the surface antigen detection reagent is used to detect the surface antigen of leukocytes. This scheme can solve the current technical problems of unsatisfactory sensitivity and specificity in the detection and diagnosis of glioma. The kit can assist imaging detection for early screening, and can also perform non-invasive screening and prediction of the progression of the disease, and realize real-time efficacy monitoring. It is of great significance for early auxiliary diagnosis of glioma patients, screening for recurrence, etc., and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor detection, and in particular to a brain glioma detection kit based on circulating tumor cells. Background Art

[0002] Gliomas are the most common primary brain tumors caused by cancerous changes in glial cells in the brain and spinal cord. According to pathology, they can be divided into astrocytomas, medulloblastomas, multiform glioblastomas, ependymomas, oligodendrogliomas, etc. Gliomas originate from glial cells in the brain and are the most common intracranial tumors, accounting for about 45% of all intracranial tumors. It ranks second among childhood malignant tumors. In the past 30 years, the incidence of primary malignant brain tumors has increased year by year, with an annual growth rate of about 1.2%, especially in the middle-aged and elderly population. According to literature reports, the annual incidence of gliomas in China is 3-8 people per 100,000 people, and the annual death toll reaches 30,000.

[0003] At present, the early screening of brain glioma mainly relies on cranial CT scans, but CT scans can only help detect intracranial lesions. In most cases, it cannot determine the nature of the lesions (benign or malignant), and may even miss some tiny lesions. Doctors usually recommend that patients undergo cranial magnetic resonance imaging (MRI), which is expensive and still difficult to diagnose. Therefore, new screening, diagnosis and detection methods are urgently needed to improve the sensitivity and specificity of brain glioma diagnosis. Summary of the invention

[0004] The present invention is intended to provide a glioma detection kit based on circulating tumor cells to solve the current technical problem of unsatisfactory sensitivity and specificity in the detection and diagnosis of glioma.

[0005] In order to achieve the above object, the present invention adopts the following technical scheme:

[0006] A circulating tumor cell-based glioma detection kit is used to detect circulating tumor cells of glioma; it comprises a transfection reagent and a surface antigen detection reagent; the transfection reagent comprises a construct, the construct contains a human telomerase reverse transcriptase promoter for regulating the construct to generate a detection signal; the surface antigen detection reagent is used to detect surface antigens of leukocytes.

[0007] The principle and advantage of this solution is that after the imaging diagnosis shows signs of brain glioma, the kit of this solution can be used for examination to assist the imaging diagnosis. In the prior art, brain glioma can only be finally diagnosed after surgical operation and relevant tests on the lesion site. It is difficult to form an accurate diagnosis conclusion through imaging methods before surgery. The use of this technical solution can increase the means of preoperative screening and diagnosis on the basis of imaging examination, and provide an important reference basis for the analysis of the necessity and urgency of surgery. In addition to preoperative judgment (early screening), the kit of this solution can also screen for recurrence in confirmed brain glioma patients, perform non-invasive screening and prediction of the progression of the disease, and perform real-time efficacy monitoring.

[0008] Circulating tumor cells (CTC) are tumor cells that break away from tumor lesions and spread into the blood. These CTC cells are the cause of malignant tumor metastasis and recurrence. CTC detection is the most potential non-invasive diagnosis and real-time efficacy monitoring method. It is generally believed in the prior art that glial cells are difficult to break through the blood-brain barrier, and there is no CTC of glioma in peripheral blood or the amount is very small, which makes it difficult to achieve effective detection. The inventor has found through a lot of research that using the kit of this scheme, glioma can be effectively diagnosed, and the kit of this scheme is of great significance for early auxiliary diagnosis of glioma patients, screening for recurrence, etc.

[0009] In summary, this scheme provides a detection kit for circulating tumor cells of brain glioma with stable detection results, high accuracy, high sensitivity, good precision, and suitable for clinical promotion. It can increase the accuracy of brain glioma diagnosis and screening, and realize non-invasive detection and prediction.

[0010] Furthermore, a brain glioma detection kit based on circulating tumor cells also includes a red blood cell lysing solution and a washing solution. The red blood cells in the sample are removed by using the red blood cell lysing solution to eliminate the influence of non-target cells on the detection. The washing solution is used to resuspend and wash the cells during the operation to remove impurities and increase the accuracy of the detection.

[0011] Further, the construct includes a fluorescent protein gene regulated by a human telomerase reverse transcriptase promoter, and the fluorescent protein gene includes at least one of a green fluorescent protein gene, a yellow fluorescent protein gene, an orange fluorescent protein gene, and a cyan fluorescent protein gene. All of the above types of fluorescent proteins can achieve an indication effect. When the fluorescent protein gene is expressed under the control of the human telomerase reverse transcriptase promoter, the cell shows a fluorescent protein signal, which is a cell that actively expresses human telomerase reverse transcriptase, i.e., a characteristic of the presence of cancer cells.

[0012] Furthermore, the construct is a recombinant oHSV1-hTERTp-eGFP virus. The recombinant oHSV1-hTERTp-eGFP virus is a virus reported in the prior patent CN103205399B (recombinant herpes simplex virus, its preparation method and application), and its deposit number is CGMCC NO.6397.

[0013] Furthermore, the surface antigen detection reagent includes an antibody to a leukocyte surface antigen labeled with a detectable marker. The antibody to a leukocyte surface antigen labeled with a detectable marker can identify leukocytes in a sample and eliminate the influence of leukocytes on the accuracy of the detection result.

[0014] Furthermore, the antibody is a CD45 antibody; the detectable marker is a Cy series fluorescent dye, an Alexa series fluorescent dye, an eFluor / Super Bright series fluorescent dye, a protein fluorescent dye, a protein and Cy series composite fluorescent dye, or a fluorescein dye. CD45 antigen is a surface characteristic antigen of leukocytes, and CD45 antibody can be used to detect leukocytes. The above fluorescent dyes are commonly used dyes in the field of cell biotechnology, and can all achieve labeling and detection of leukocytes.

[0015] Furthermore, the Cy series fluorescent dyes include CY-7; the protein fluorescent dyes include PE, APC, and PerCP; the protein and Cy series composite fluorescent dyes include PE-CY5, PerCP-CY5.5, and APC-CY7; and the fluorescein dyes include FITC. The above specific types of dyes are common dyes in the art, stable in nature, and easy to obtain.

[0016] Furthermore, the recombinant oHSV1-hTERTp-eGFP virus is used at MOI=1; the amount of CD45 is 30-50ul per 4ml blood sample; after the blood sample is anticoagulated and centrifuged, the plasma is discarded and the cell part is retained, and red blood cell lysis solution is added to the cell part, and the amount of red blood cell lysis solution is 10-15 times the volume of the cell part. The virus infection efficiency of this scheme is high, and the cells in the sample to be tested can be transfected at a ratio of MOI=1. In actual operation, adding 30-50ul can effectively label white blood cells and avoid mislabeling of other types of cells.

[0017] Furthermore, the red blood cell lysing solution is Tris-NH4Cl lysing solution or ACK lysing solution; the washing solution is phosphate buffer. The washing solution and red blood cell lysing solution are both conventionally implemented in the prior art, easy to obtain and have ideal effects.

[0018] Furthermore, a circulating tumor cell-based glioma detection kit is used in the preparation of a glioma diagnostic preparation, wherein the kit is used to detect cells that do not have CD45 antigen on their surface and express GFP fluorescence; if the number of the cells is greater than 1, the test result is judged to be positive; if the number of the cells is less than or equal to 1, the test result is judged to be negative; the number of the cells is detected by flow cytometry, and the FSC parameters and SSC parameters of the flow cytometer are adjusted to a level greater than that of the lymphocyte subpopulation.

[0019] The inventor has detected the peripheral blood of nearly 100 clinical patients with different types of glioma and healthy volunteers, and the data show that patients and normal people can be effectively distinguished, and there are obvious differences in CTC values. The inventor has then made use of the above new discovery and realized the in vitro diagnosis and detection of glioma using the kit of the present invention. The kit of this scheme can be used for the diagnosis of glioma by peripheral blood auxiliary clinical. In addition, by testing a large number of clinical samples, the threshold value (i.e., the interpretation result in Table 4) for glioma detection has been determined, so that the sensitivity and specificity of detection are greatly improved. In addition, this scheme optimizes flow cytometry detection, and the FSC parameters and SSC parameters of the flow cytometer are adjusted to a level greater than the lymphocyte subpopulation, which can eliminate the influence of regions such as debris on the test results and improve the detection accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a flow cytometry analysis diagram before optimization of Example 1 of the present invention.

[0021] Figure 2 This is a flow cytometry analysis diagram after optimization of Example 1 of the present invention.

[0022] Figure 3 This is the linear range detection result of Experimental Example 1 of the present invention.

[0023] Figure 4 This is the ROC analysis result of Experimental Example 2 of the present invention.

[0024] Figure 5 This is the CTC statistical result of brain glioma in Experimental Example 3 of the present invention. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial sources.

[0026] Embodiment 1:

[0027] 1. Materials and Methods

[0028] The composition of the kit is shown in Table 1, the instruments to be used with the kit are shown in Table 2, and the raw materials of the kit and the reagents required for the test are shown in Table 3.

[0029] Table 1: Kit composition

[0030]

[0031] Table 2: Instruments used with the kit

[0032] name Specifications Manufacturer Flow cytometry FACSCantoⅡ Becton Dickinson (USA) Biological Safety Cabinets HDL Beijing Donglian Har Instrument Manufacturing Co., Ltd. <![CDATA[CO2 Incubator]]> MCO-15AC Sanyo Electric Co., Ltd.(Japan) Analytical Balance TE214S Sartorius pH Meter pHS-3 Series Shanghai Precision Instruments Co., Ltd.

[0033] Table 3: Kit materials and reagents

[0034] name Manufacturer CD45 Antibody eBioscience <![CDATA[KHCO3]]> Sinopharm Chemical Reagent Co., Ltd. NH4CL Sinopharm Chemical Reagent Co., Ltd. <![CDATA[EDTA-Na2]]> Sinopharm Chemical Reagent Co., Ltd. KCl Sinopharm Chemical Reagent Co., Ltd. NaCl Sinopharm Chemical Reagent Co., Ltd. <![CDATA[Na2HPO4]]> Sinopharm Chemical Reagent Co., Ltd. <![CDATA[KH2PO4]]> Sinopharm Chemical Reagent Co., Ltd.

[0035] In this technical solution, the recombinant oHSV1-hTERTp-eGFP virus is a virus reported in the prior patent CN103205399B (recombinant herpes simplex virus, its preparation method and application), and its deposit number is CGMCC NO.6397. The titer of the virus in the kit is ≥5x10 7 PFU / ml.

[0036] CD45 antibodies can be labeled in a variety of ways, that is, a variety of detectable markers can be used, including at least one of Cy series fluorescent dyes, Alexa series fluorescent dyes, eFluor / Super Bright series fluorescent dyes, protein fluorescent dyes, protein and Cy series composite fluorescent dyes, and fluorescein dyes. More specifically, Cy series fluorescent dyes can include CY-7; protein fluorescent dyes include PE, APC, and PerCP; protein and Cy series composite fluorescent dyes include PE-CY5, PerCP-CY5.5, and APC-CY7; and fluorescein dyes include FITC.

[0037] The washing solution is a conventional phosphate buffered saline (PBS) in the prior art. In this embodiment, the specific preparation method is as follows:

[0038] Weigh 7.9g NaCl, 0.2g KCl, 0.24g KH2PO4, 1.8g K2HPO4, adjust the pH value of the solution to 7.4, and finally add distilled water to make up to 1L. The preparation of PBS buffer and its application in biological preparations belong to conventional means in the prior art. Its composition and dosage, pH and concentration can be adaptively adjusted according to actual conditions. The use of commercial products can achieve the purpose of this technical solution.

[0039] Red blood cell lysis solution is also a conventional reagent in the prior art for lysing and removing red blood cells to eliminate the interference of red blood cells on sample detection. Its main principle is to adjust the osmotic pressure of red blood cells to cause lysis, as long as the above purpose can be achieved. In this embodiment, the specific preparation method is as follows:

[0040] KHCO3 1.0g, NH4CL 8.3g, EDTA-Na2 0.037g, adjust the pH value of the solution to 7.2, and finally add distilled water to make up to 1L. The preparation of red blood cell lysis solution and its application in lysing red blood cells belong to conventional means in the prior art. Its composition and dosage, pH and concentration can be adaptively adjusted according to actual conditions. The use of commercial products can achieve the purpose of this technical solution.

[0041] 2. Testing process

[0042] (1) Sample collection: 4 mL of venous blood was collected and placed in an EDTA anticoagulant tube.

[0043] (2) Take out the components except the antibody from Kit A and equilibrate at room temperature for 30 minutes.

[0044] (3) Centrifuge the blood sample at 700 g for 5 minutes to remove the plasma.

[0045] (4) After removing the plasma, transfer all the remaining cells to a 50 ml centrifuge tube, add red blood cell lysis solution (volume ratio 1:12), mix well and let stand for 10 minutes. In actual operation, the amount of red blood cell lysis solution added can be 10-15 times the volume of the cell part.

[0046] (5) Centrifuge at 500 g for 5 min and discard the supernatant.

[0047] (6) After centrifugation, discard the top layer of liquid and resuspend the cells in 6 mL of washing solution.

[0048] (7) After centrifugation, discard the supernatant, resuspend and wash the cells with 1 mL of serum-free cell culture medium (commercial 1640 culture medium), mix gently and centrifuge at 700 g for 5 minutes. Then discard the supernatant, add 1 mL of cell culture medium (specifically, 1640 culture medium containing 10% fetal bovine serum), resuspend the cells, and spread 1 mL of the cell suspension into a six-well plate.

[0049] (8) Transfection: Take out the transfection reagent (the transfection reagent is the recombinant oHSV1-hTERTp-eGFP virus), blow the transfection reagent several times with a pipette to mix it evenly, add 100 μl of the transfection reagent to each well to infect the cells, and shake gently. Incubate in a 37°C carbon dioxide cell culture incubator (5%) for 1 hour, and then add 1 mL of complete cell culture fluid (i.e., 1640 culture medium containing 10% fetal bovine serum) to each well. Continue to incubate in the cell culture incubator, and collect the cells after 16-24 hours. In this step, the recombinant oHSV1-hTERTp-eGFP virus is used at MOI=1. MOI is (Multiplicity of Infection), which means the ratio of the number of viruses to the number of cells during infection.

[0050] (9) Collect cells: pipette the cells in the six-well plate into a 5 mL centrifuge tube and centrifuge at 700 g for 5 minutes.

[0051] (10) Discard the supernatant, add 3 mL of washing buffer to each centrifuge tube to resuspend the washed cells, and centrifuge at 700 g for 5 minutes.

[0052] (11) Antibody labeling of cells: Add 30 μl of CD45 antibody to the cell suspension to label white blood cells. In actual operation, adding 30-50 μl can effectively label white blood cells and avoid mislabeling of other types of cells.

[0053] (12) Washing: After incubation, add 3 mL of washing solution to the centrifuge tube and mix well, and centrifuge at 700 g for 5 minutes. After centrifugation, discard the supernatant, add 0.5 mL of washing solution and resuspend to ensure that the cells are completely dispersed and there are no other insoluble debris. Then use a flow cytometer to detect CD45- / GFP+ cells, that is, cells that do not have CD45 antigen on the surface and express GFP fluorescence, and count the number of cells. The judgment criteria for negative and positive are shown in Table 4. Among them, when detecting by flow cytometry, adjust the FSC / SSC parameters to be greater than "lymphocyte subsets".

[0054] Table 4: Test result interpretation

[0055] Test results Reference interval Result determination Number of CD45(-) / GFP(+) cells >1 Positive Number of CD45(-) / GFP(+) cells <＝1 Negative

[0056] In the above technical scheme, the processes of anticoagulation of venous blood, extraction of cell parts in venous blood, lysis and removal of red blood cells, virus transfection, antibody labeling of cells, etc. are all conventional technical schemes in this field, and some parameters can be adjusted according to actual needs. The above operation process is the most optimized standard operation step, which can obtain the most ideal test results.

[0057] Since this technical solution is aimed at detecting brain glioma cells, brain glioma cells need to break through the blood-brain barrier to enter the peripheral blood. Compared with other solid tumors, their content in peripheral blood is quite different, and brain glial cells differ from ordinary cells in morphology and composition. This leads to the need to redefine the detection conditions when performing flow cytometry on samples transfected with viruses and labeled surface antibodies to ensure good detection results. This solution optimizes the previous flow cytometry methodology by combining the characteristics of glioma cells themselves, and directly eliminates the influence of debris and other areas on the detection results. See the flow cytometry analysis diagram before optimization. Figure 1 , the optimized flow cytometry analysis diagram is shown in Figure 2 Before optimization, the FSC / SSC parameters were not adjusted to a level greater than the "lymphocyte subsets". After optimization, the FSC / SSC parameters were adjusted to a level greater than the "lymphocyte subsets". The image obtained after optimization excluded the influence of debris and other areas on the detection results, and only the P1 area existed in the image.

[0058] In addition, by testing a large number of clinical samples (106 cases), the study determined the threshold for glioma detection (i.e., the interpretation results in Table 4), which greatly improved the sensitivity and specificity of the detection. The detection threshold in the prior art is usually "0". For example, the prior patent CN109679928B (recombinant herpes simplex virus, kit and its use) detects prostate cancer CTC, and the detection threshold is "0". However, for the special case of glioma CTC, a detection threshold of "1" is required to obtain accurate detection results. If a detection threshold of "0" is used for glioma CTC, a large number of false positives will occur.

[0059] Example 2

[0060] Experimental Example 1: Performance Analysis of the Kit

[0061] 1. Recovery rate

[0062] a) Take 50 positive control cells (derived from U373 human glioma cell line, purchased from Peking Union Medical College Cell Resource Center) and 100 positive control cells and add them to 2.5x10 6In the negative reference of leukocytes (leukocytes isolated from healthy human peripheral blood), 3 samples of each concentration of positive control were prepared, and each sample was measured once as a recovery sample. The sample without positive control cells was prepared in the same way as the basic sample, and the recovery rate was calculated according to formula (1). The recovery rate was greater than 60% (the conventional flow cytometer can be used to achieve the detection purpose, and there is no special requirement for its model. It only needs to have dual lasers, such as 488, 640 dual laser tubes). More specific experimental data are: 50 positive control cell groups were measured three times, and the recovery rates were 62%, 60%, and 64% respectively; 100 positive control cell groups were measured three times, and the recovery rates were 63%, 66%, and 69% respectively.

[0063]

[0064] b) Five accuracy clinical samples (cancer patients diagnosed at the Cancer Hospital of the Chinese Academy of Medical Sciences) were tested according to the instructions of the test kit (tested according to the above testing process), and the results were all positive.

[0065] 2. Precision

[0066] Take 50 and 100 positive control cells respectively and add them into 2.5×10 6 The negative reference of leukocytes was tested 10 times, and the mean value (Mean) and standard deviation (SD) of the number of positive control cells detected in the 10 tests (detected according to the above test process) were calculated. The coefficient of variation (CV) was calculated according to formula (2) and formula (3), and the results were all less than 10%. More specifically, the 50 positive control cell groups and the 100 positive control cell groups were both 5%.

[0067]

[0068] CV = (SD / mean value) × 100% Formula (3)

[0069] Where: x is the detection value of the number of cells of the positive quality control product; n is the number of detections.

[0070] 3. Linear

[0071] 20, 50, 100, 150 and 200 positive control cells were added to 2.5×10 6 The negative reference of leukocytes was tested, and the amount of positive control cells added (20, 50, 100, 150 and 200) was used as the independent variable. The mean of the positive control cell test results was used as the dependent variable for linear fitting. The correlation coefficient should not be less than 0.9500. The analysis results are as follows Figure 1The linear range test results are as follows: 20 cells test results (true values) are 11 and 12; 50 cells test results are 25 and 34; 100 cells test results are 57 and 49; 150 cells test results are 90 and 82; 200 cells test results are 122 and 122. Then perform linear fitting based on the above data to obtain the following: Figure 3 The curve shown has a correlation coefficient of 0.993, which meets the application requirements.

[0072] Experimental Example 2: Testing of healthy volunteers and patients with brain glioma

[0073] 1. This kit was used to study samples from 100 healthy volunteers. The experimental results are shown in Table 5. The detection values ​​for healthy people were all 0, proving that the positive detection rate of this test kit is extremely low.

[0074] Table 5: Circulating tumor cell (CTC) detection results

[0075]

[0076] 2. This kit was used to study samples from 50 patients with glioma. The experimental results are shown in Table 6. The average value for patients with glioma was 2.3, and the detection value range was 0-8.

[0077] Table 6: Circulating tumor cell (CTC) detection results

[0078]

[0079] 3. Sensitivity and specificity analysis

[0080] ROC analysis was performed on the test results of the above healthy people and glioma patients. The analysis results are as follows Figure 4 As shown, Figure 4 The results showed that when the detection value was >0, the sensitivity of the detection was 80% and the specificity was 100%.

[0081] Experimental Example 3: CTC detection results of brain glioma cells

[0082] Since gliomas occur in the brain, and the presence of the blood-brain barrier makes people believe that circulating tumor cells (CTCs) disseminated by gliomas will not be detected in the peripheral blood. The present invention tested 4 ml of peripheral blood from nearly 100 clinical patients with different types of gliomas and healthy volunteers, and counted the number of glioma CTCs in 4 ml of peripheral blood. See Table 7 for experimental case statistical information, where A is astrocytoma, O is oligodendroglioma, AO is anatomic cell tumor, OA is oligodendroglioma, AOA is oligodendroglioma, and GBM is glioblastoma cell tumor. The results of the tests on these clinical samples can be found in Figure 5. Because gliomas occur in the brain, and the presence of the blood-brain barrier makes people believe that circulating tumor cells (CTCs) disseminated by gliomas will not be detected in the peripheral blood. The present invention tested 4 ml of peripheral blood from nearly 100 clinical patients with different types of gliomas and healthy volunteers (using the kit of this solution and the detection process of Example 1). The data showed that the patients and normal people can be effectively distinguished, and there are obvious differences in CTC values. This shows that the present invention can be used as an in vitro diagnostic reagent to assist in the clinical diagnosis of gliomas through peripheral blood.

[0083] Table 7: Experimental case statistics

[0084]

[0085] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. Application of a circulating tumor cell-based glioma detection kit in the preparation of a glioma diagnostic system, characterized in that: The brain glioma diagnosis system comprises a brain glioma diagnosis preparation and a flow cytometer; the brain glioma detection kit based on circulating tumor cells is used to detect circulating tumor cells of brain glioma; it comprises a transfection reagent and a surface antigen detection reagent; the transfection reagent comprises a construct, the construct comprises a human telomerase reverse transcriptase promoter for regulating the construct to generate a detection signal; the construct comprises a fluorescent protein gene regulated by the human telomerase reverse transcriptase promoter, the fluorescent protein gene comprises a green fluorescent protein gene; the surface antigen detection reagent is used to detect the surface antigen of leukocytes; the surface antigen detection reagent comprises an antibody to the leukocyte surface antigen labeled with a detectable marker; the antibody is a CD45 antibody; The kit is used to detect cells that do not have CD45 antigen on their surface and express GFP fluorescence; if the number of the cells is greater than 1, the test result is determined to be positive; if the number of the cells is less than or equal to 1, the test result is determined to be negative; The flow cytometer is used to detect the number of the cells, and the FSC parameter and SSC parameter of the flow cytometer are both adjusted to a level greater than that of the lymphocyte subpopulation.

2. The use of a circulating tumor cell-based glioma detection kit according to claim 1 in the preparation of a glioma diagnostic system, characterized in that: The circulating tumor cell-based glioma detection kit also includes a red blood cell lysis solution and a washing solution.

3. The use of a circulating tumor cell-based glioma detection kit in the preparation of a glioma diagnostic system according to claim 2, characterized in that: The red blood cell lysing solution is Tris-NH4Cl lysing solution or ACK lysing solution; the washing solution is phosphate buffer.

4. The use of a circulating tumor cell-based glioma detection kit according to claim 1 in the preparation of a glioma diagnostic system, characterized in that: The construct is a recombinant oHSV1-hTERTp-eGFP virus.

5. The use of a circulating tumor cell-based glioma detection kit in the preparation of a glioma diagnostic system according to claim 4, characterized in that: The recombinant oHSV1-hTERTp-eGFP virus is used at MOI=1; the amount of the CD45 antibody is 30-50ul per 4ml blood sample; after the blood sample is anticoagulated and centrifuged, the plasma is discarded and the cell portion is retained, and red blood cell lysis solution is added to the cell portion, and the amount of red blood cell lysis solution is 10-15 times the volume of the cell portion.

6. Use of a circulating tumor cell-based glioma detection kit according to claim 1 in preparing a glioma diagnostic system, characterized in that: The detectable marker includes at least one of Cy series fluorescent dyes, Alexa series fluorescent dyes, eFluor / Super Bright series fluorescent dyes, protein fluorescent dyes, protein and Cy series composite fluorescent dyes and fluorescein dyes.

7. Use of a circulating tumor cell-based glioma detection kit according to claim 6 in preparing a glioma diagnostic system, characterized in that: The Cy series fluorescent dyes include CY-7; the protein fluorescent dyes include PE, APC and PerCP; the protein and Cy series composite fluorescent dyes include PE-CY5, PerCP-CY5.5 and APC-CY7; the fluorescein dyes include FITC.

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