CN113687068A - Tumor single cell double-fluorescence labeling detection kit, preparation method and application thereof - Google Patents

Tumor single cell double-fluorescence labeling detection kit, preparation method and application thereof Download PDF

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CN113687068A
CN113687068A CN202110972450.7A CN202110972450A CN113687068A CN 113687068 A CN113687068 A CN 113687068A CN 202110972450 A CN202110972450 A CN 202110972450A CN 113687068 A CN113687068 A CN 113687068A
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tumor
reagent
detection kit
single cell
cells
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朱乃硕
梁雨来
朱嗣博
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Jiangsu Yaoyanda Technology Development Co ltd
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Abstract

The invention relates to a tumor single cell dual-fluorescence labeling detection kit, a preparation method and application thereof, wherein the detection kit comprises: the reagent A comprises a deoxyglucose-2- (7-nitrobenzo-2-oxa-1, 3-diazole-4-amino) solution with the concentration of more than 10mg/mL, which is prepared from a buffer solution, and the reagent B comprises a Rox-EpCAM-mAb freeze-dried powder solution with the concentration of more than 1 mg/mL. The invention has dual specificity to indicate tumor cells, the circulating tumor cells marked by the detection kit can be accurately identified and separated by a tumor single cell counting and separating device system (TuSEP), the high-efficiency and accurate separation and acquisition of the circulating tumor cells are ensured, and a foundation is laid for further carrying out the personalized and accurate treatment of tumor patients.

Description

Tumor single cell double-fluorescence labeling detection kit, preparation method and application thereof
Technical Field
The invention relates to the technical field of biological medicines, in particular to a tumor single cell double-fluorescence labeling detection kit, a preparation method and application thereof.
Background
The tumors seriously threaten the health of human beings, the main treatment means at present comprise operations, radiotherapy, chemotherapy, immunobiotherapy and the like, then the treatment modes all need early accurate diagnosis to be accurately treated, most cancer patients are discovered to be in a late stage and cannot be radically treated due to the lack of accurate diagnosis technology for the early stage of the tumors, and the 5-year survival rate is very low.
At present, the diameter of the tumor can reach more than 1cm, and can be found by physical methods such as CT, nuclear magnetic resonance and the like, but the confirmed diagnosis still needs pathological observation of tumor tissue cells after biopsy and operation to confirm the diagnosis, even if the diagnosis does not reach the molecular level; due to the high complexity of tumors and the high heterogeneity of cells and their genes, it is particularly important for the personalized and precise treatment of tumors.
Tumors have the characteristics of unlimited growth and metastasis, and accurate treatment aiming at individuation is difficult to be carried out in time due to the lack of an early accurate diagnosis method, so that most (more than 90 percent) tumor patients die of tumor metastasis. After the tumor growth breaks through the basement membrane of early in situ cancer, it is presumed that each gram of tumor tissue can release nearly 10 ten thousand tumor cells (circulating tumor cells, CTCs) into blood and surrounding tissue fluid every day, which provides a possible pathological material and material basis for early accurate diagnosis, however, each milliliter of blood has billions of cells, each milliliter of cells after being diluted in a large amount has only a few tumor cells, which are difficult to separate and detect, and which is a medical difficulty and a scientific problem facing all over the world.
Disclosure of Invention
In order to solve the technical problems, the invention provides a tumor single cell dual-fluorescence labeling detection kit, a preparation method and application thereof, and the kit has the advantages of accurately separating and counting tumor cells.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a tumor single cell dual-fluorescence labeling detection kit comprises: the reagent A comprises a deoxyglucose-2- (7-nitrobenzo-2-oxa-1, 3-diazole-4-amino) solution with the concentration of more than 10mg/mL, which is prepared from a buffer solution, and the reagent B comprises a Rox-EpCAM-mAb freeze-dried powder solution with the concentration of more than 1 mg/mL.
As a preferred scheme of the invention, the buffer solution in the reagent A is phosphate buffer solution, and the Rox-EpCAM-mAb freeze-dried powder solution is prepared from 50% glycerol.
As a preferred embodiment of the invention, the Rox-EpCAM-mAb lyophilized powder comprises 0.01M phosphate buffer, 1% BSA and 0.1% sodium azide.
In another aspect, the present invention further provides a method for preparing the detection kit according to any one of the above technical schemes, comprising:
weighing 10mg of deoxyglucose-2- (7-nitrobenzo-2-oxa-1, 3-diazole-4-amino) and dissolving the deoxyglucose-2- (7-nitrobenzo-2-oxa-1, 3-diazole-4-amino) by using 1mL of phosphate buffer solution to prepare a reagent A with the final concentration of 10 mg/mL;
dissolving 1mg of Rox-EpCAM-mAb freeze-dried powder in 1mL of 50% glycerol solution to prepare a reagent B with the final concentration of 1 mg/mL;
and storing the reagent A and the reagent B in a refrigerated way at the temperature of-20 ℃.
As a preferred embodiment of the invention, the Rox-EpCAM-mAb lyophilized powder comprises 0.01M phosphate buffer, 1% BSA and 0.1% sodium azide.
On the other hand, the invention also provides application of the detection kit in any technical scheme in fluorescence detection of human tissues and blood circulation tumor cells.
As a preferred aspect of the present invention, the application includes:
taking part of primary focus/metastatic focus tissues of human or animal with tumor or extracting 3-4mL of peripheral blood, digesting the tumor tissues into single cell suspension by pancreatin for later use, and adding anticoagulant into the peripheral blood for later use;
adding the reagent A and the reagent B into a single cell suspension of human tumor tissue or a peripheral blood sample in a ratio of 1:100 and a ratio of 1:1000, placing the single cell suspension or the peripheral blood sample into a cell culture box containing 5% carbon dioxide and at the temperature of 37 ℃ in a dark environment for culturing and staining for 0.5 to 1 hour, then adding erythrocyte lysate with the volume of 3 to 5 times of the sample volume, gently blowing and uniformly mixing, treating at room temperature for 2 minutes, centrifuging 500g for 5 minutes, removing supernatant, adding 1mL of phosphate buffer solution for resuspending cells, and detecting the fluorescent staining condition of the cells in the human tumor tissue or the peripheral blood under a confocal microscope.
In a preferred embodiment of the present invention, the method further comprises the step of mixing the reagent a and the reagent B in a ratio of 10: 1, taking 25-50 mu L of the mixture, carrying out micro-intravenous injection on a tumor-bearing mouse, and carrying out fluorescence living body imaging observation on the mouse after 3 hours.
On the other hand, the invention also provides application of the detection kit of any technical scheme in the identification of gene mutation and expression information by matching with a tumor single cell gene sequencing technology.
As a preferred embodiment of the invention, the application is implemented based on single tumor cells separated after human tissues and blood circulation tumor cells are labeled by staining of a detection kit.
In conclusion, the invention has the following beneficial effects:
the embodiment of the invention provides a tumor single cell dual-fluorescence labeling detection Kit, a preparation method and application thereof, wherein a main component of the detection Kit (TuSTA Kit) is deoxyglucose-2- (7-nitrobenzo-2-oxa-1, 3-diazole-4-amino) which is a green fluorescence labeling glucose derivative (FG) and has small molecular weight and is easily and efficiently absorbed by tumor cells, and the other main component Rox-EpCAM-mAb is an EpCAM specific IgG monoclonal antibody molecule marked by a red fluorescence group ROX and is a tumor cell specific labeling molecule, and the detection Kit and the Rox-EpCAM-mAb can indicate the tumor cells when combined for use and have dual specificity; the circulating tumor cells marked by the detection kit can be accurately identified and separated by a tumor single cell counting and separating device system (TuSEP), so that the high-efficiency accurate separation and acquisition of the circulating tumor cells are ensured; the 3TS system composed of the detection kit TuSTA, TuSEP and TuSEQ realizes high-efficiency detection aiming at early circulating tumor cells from stages of identification, separation, acquisition and sequencing analysis, establishes a set of complete tumor precision detection system, and lays a foundation for further carrying out personalized precision treatment on tumor patients.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic molecular structure diagram of Rox-EpCAM-mAb in example two of the present invention.
FIG. 2 is a graph showing the results of dual fluorescence labeling of tumor cells using the detection kit in the third embodiment of the present invention.
FIG. 3 is the result of in vivo fluorescence observation of tumors in the fluorescence-labeled animals (mice) in the third example of the present invention.
FIG. 4 is a flowchart illustrating the operation of the tumor single-cell counting and separating device according to the fourth embodiment of the present invention.
FIG. 5 is a diagram showing the results of TuSTA Kit detection of circulating tumor cells isolated by a TuSEP device according to the fourth embodiment of the present invention.
FIG. 6 is a chart of the counts of the detection of circulating pancreatic cancer cell (CTC) signals after TuSEP analysis of TuSTA markers in the fourth embodiment of the present invention.
Fig. 7 is a flowchart illustrating operation of TuSEQ technology platform according to an embodiment of the present invention.
FIG. 8 shows the result of TuSEQ sequencing for identifying colon cancer CTC and high-frequency gene mutation sites in example V of the present invention; wherein, A is an expression quantitative chart, and B is gene mutation site information.
FIG. 9 shows the result of TuSEQ sequencing for identifying CTC of pancreatic cancer and high-frequency gene mutation sites in example V of the present invention; wherein, A is an expression quantitative chart, and B is gene mutation site information.
FIG. 10 shows the effect of personalized vaccine for treating colon cancer tumor in the fifth embodiment of the present invention; wherein, the red line of the A picture and the bottom line of the B picture show that the tumor volume is obviously reduced after the individual tumor gene vaccine is used for immunization.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example one
A tumor single cell dual-fluorescence labeling detection kit comprises: the reagent A comprises a deoxyglucose-2- (7-nitrobenzo-2-oxa-1, 3-diazole-4-amino) solution with the concentration of more than 10mg/mL, which is prepared from a buffer solution, and the reagent B comprises a Rox-EpCAM-mAb freeze-dried powder solution with the concentration of more than 1 mg/mL.
The buffer solution in the reagent A is phosphate buffer solution, the Rox-EpCAM-mAb freeze-dried powder solution is prepared from 50% glycerol, and the Rox-EpCAM-mAb freeze-dried powder comprises 0.01M phosphate buffer solution, 1% BSA and 0.1% sodium azide.
The embodiment of the invention provides a tumor single cell dual-fluorescence labeling detection Kit, a preparation method and application thereof, wherein a main component of the detection Kit (TuSTA Kit) is deoxyglucose-2- (7-nitrobenzo-2-oxa-1, 3-diazole-4-amino) which is a green fluorescence labeling glucose derivative (FG) and has small molecular weight and is easily and efficiently absorbed by tumor cells, and the other main component Rox-EpCAM-mAb is an EpCAM specific IgG monoclonal antibody molecule marked by a red fluorescence group ROX and is a tumor cell specific labeling molecule, and the detection Kit and the Rox-EpCAM-mAb can indicate the tumor cells when combined for use and have dual specificity; the circulating tumor cells marked by the detection kit can be accurately identified and separated by a tumor single cell counting and separating device system (TuSEP), so that the high-efficiency accurate separation and acquisition of the circulating tumor cells are ensured; the 3TS system composed of the detection kit TuSTA, TuSEP and TuSEQ realizes high-efficiency detection aiming at early circulating tumor cells from stages of identification, separation, acquisition and sequencing analysis, establishes a set of complete tumor precision detection system, and lays a foundation for further carrying out personalized precision treatment on tumor patients.
Example two
A method of making the test kit of example one, comprising:
weighing 10mg of deoxyglucose-2- (7-nitrobenzo-2-oxa-1, 3-diazole-4-amino) and dissolving by using 1mL of phosphate buffer solution to prepare a reagent A with the final concentration of 10 mg/mL;
step two, dissolving 1mg of Rox-EpCAM-mAb freeze-dried powder in 1mL of 50% glycerol solution to prepare a reagent B with the final concentration of 1mg/mL, wherein the Rox-EpCAM-mAb freeze-dried powder comprises 0.01M phosphate buffer, 1% BSA and 0.1% sodium azide, and the molecular structure diagram of the Rox-EpCAM-mAb freeze-dried powder is shown in figure 1;
and step three, refrigerating and storing the reagent A and the reagent B at the temperature of-20 ℃.
EXAMPLE III
An application of the detection Kit (TuSTA Kit) in human tissue and blood circulation tumor cell fluorescence detection specifically comprises:
taking part of primary focus/metastatic focus tissues of human or animal with tumor or extracting 3-4mL of peripheral blood, digesting the tumor tissues into single cell suspension by pancreatin for later use, and adding anticoagulant into the peripheral blood for later use;
adding the reagent A and the reagent B into a single cell suspension of human tumor tissue or a peripheral blood sample in a ratio of 1:100 and a ratio of 1:1000, placing the single cell suspension or the peripheral blood sample into a cell culture box containing 5% carbon dioxide and at the temperature of 37 ℃ in a dark environment for culturing and staining for 0.5 to 1 hour, then adding erythrocyte lysate with the volume of 3 to 5 times of the sample volume, gently blowing and uniformly mixing, treating at room temperature for 2 minutes, centrifuging 500g for 5 minutes, removing supernatant, adding 1mL of phosphate buffer solution for resuspending cells, and detecting the fluorescent staining condition of the cells in the human tumor tissue or the peripheral blood under a confocal microscope.
Deoxyglucose-2- (7-nitrobenzo-2-oxa-1, 3-diazole-4-amino) is a green fluorescence labeled glucose derivative (FG), Rox-EpCAM-mAb is an EpCAM specific IgG monoclonal antibody molecule labeled by a red fluorescence group ROX, and as shown in a result of a graph 1, 82 cells singly labeled by FG in 89 cells in total in a tumor tissue lysate (green fluorescence, detection rate of 92.1%), 87 cells singly labeled by Rox-EpCAM-mAb (red fluorescence, detection rate of 97.8%) and 78 cells shared by two colors (accuracy rate of 95.1%); while 118 cells out of 128 total cells in the peripheral blood sample labeled with FG green fluorescence (green fluorescence, detection rate 92.2%), 124 cells labeled with Rox-EpCAM-mAb red fluorescence (red fluorescence, detection rate 96.9%), and 110 cells in common in both colors (accuracy rate 93.2%), indicating that TuSTA Kit has high specificity and accuracy in labeling circulating tumor cells.
Further, this embodiment further includes the steps of mixing reagent a and reagent B in a ratio of 10: mixing at a ratio of 1, taking 25-50 μ L, injecting tumor-bearing mice into micro vein, and observing the mice by fluorescence living body imaging after 3h, wherein the result is shown in figure 3.
Example four
An application of the detection kit in the first embodiment in combination with a tumor single cell counting and separating device for counting and separating tumor cells.
Wherein, the tumor single cell counting and separating device (TuSEP) mainly comprises: the device comprises a control box, a micro-injection pump, a linear guide rail, an electromagnet, a chip position adjusting device, a micro-fluidic sorting chip, a micro-fluidic detection chip, a power-off button, a control panel and a fluorescence detection device.
The cell sap stained by the reagent A and the reagent B of the detection kit is further precisely counted and separated by a tumor single cell counting and separating device (TuSEP), and the specific operation steps of Circulating Tumor Cell (CTC) detection and sorting by the TuSEP are shown in figure 4 and comprise the following steps:
A. a power plug on a control box in the dual-channel circulating tumor cell detector is inserted into a 220V network power supply, a main power supply (green boat-shaped) switch on the control box is turned on, and meanwhile, an indicator lamp of the main power supply switch is turned on.
B. And installing the microfluidic sorting chip and the microfluidic detection chip on a chip position adjusting device.
Note: the micro-fluidic chip mounting method comprises the following steps:
steel needles connected to a tygon hose were inserted into the sample outlet and sample inlet of the microfluidic chip.
C. And opening the upper computer control software and the CCD camera software, and simultaneously adjusting the position of the microfluidic detection chip by using the chip position adjusting device to ensure that the chip flow channel clearly images in an imaging interface of the CCD camera software.
Note: the method for adjusting the position of the chip comprises the following steps:
the position of the chip is adjusted by using a left fine adjustment knob and a right fine adjustment knob, so that a flow channel of the chip appears in a software imaging interface of the CCD camera; and the upper and lower fine adjustment knobs are used for adjusting the focal length from the chip to the fluorescence detection device, so that the optimal imaging effect is achieved.
D. And (3) filling purified water into the centrifuge tube, performing a work flow by using the purified water, and checking whether each part of the equipment works normally.
E. Preparing a sample to be tested, and filling the sample into a centrifugal tube.
F. Starting software, firstly registering sample and patient information; then entering a CTCs sorting module, setting parameters such as sample introduction speed, sample capacity and the like in an interface, and clicking a work starting button to start CTCs sorting; entering a CTCs detection module after sorting is finished, setting parameters such as sample introduction speed, sample capacity and the like in an interface, and clicking a start working button to start CTCs detection; and after the CTCs are detected, entering report printing and printing a patient detection report.
As shown by the results of fig. 5 and 6: after the cells are stained by the detection kit, the analysis of a tumor single cell counting and separating device shows that 89 cells (green fluorescence, the detection rate is 92.7%) marked by deoxyglucose-2- (7-nitrobenzo-2-oxa-1, 3-diazole-4-amino) in 96 total cells in tumor tissues, 92 cells (red fluorescence, the detection rate is 95.8%) marked by Rox-EpCAM-mAb, and 82 cells (the accuracy rate is 92.1%) shared by two colors are contained in the cells; and 145 cells (green fluorescence, the detection rate is 92.9%) marked by deoxyglucose-2- (7-nitrobenzo-2-oxa-1, 3-diazole-4-amino) in 156 total cells in peripheral blood, 151 cells (red fluorescence, the detection rate is 96.8%) marked by Rox-EpCAM-mAb, and 134 cells (the accuracy rate is 92.4) shared by two colors, which indicates that the tumor cells marked by the TuSTA Kit can be accurately captured and counted by the TuSEP system and then sorted (separated), and are used for cell property identification and gene sequencing analysis by the next tumor single cell gene sequencing technology (TuSEQ technical platform).
EXAMPLE five
The application of the detection kit of the embodiment I in cooperation with a tumor single cell gene sequencing technology (TuSEQ technology platform) to identify gene mutation and expression information is implemented on the basis of tumor single cells separated after human tissues and blood circulation tumor cells are labeled by staining of the detection kit.
As shown in fig. 7, fig. 7 shows an operation flowchart of TuSEQ technology platform, and the specific operation implementation steps are as follows:
1. single cell reverse transcription
About 0.3 μ L of CTCs was pipetted using a capillary tube, 2 μ L of red blood cell lysate was added, and single cell reverse transcription conditions 1 were prepared according to the following table:
Figure BDA0003226353900000081
during treatment, the mixture was placed at 72 ℃ for reaction for 3min (the temperature was adjusted to 72 ℃ by a hot lid), and the mixture was placed on ice after completion of the reaction.
Then, the single cell reaction system is mixed evenly according to the following table and added into the single cell reverse transcription condition 1 to obtain a single cell reverse transcription condition 2,
Figure BDA0003226353900000082
the 10. mu.L total volume of the reaction was placed in and the single cell reverse transcription reaction was rapidly initiated.
The PCR instrument (Biorad CFX96 PCR) was turned on, the hot lid temperature was closed, and the:
1) reacting at 42 ℃ for 90 minutes;
2) (reaction at 50 ℃ for 2 min + reaction at 42 ℃ for 2 min) 10 times;
3) reacting for 15 minutes at 70 ℃;
4) the reaction was terminated at 4 ℃.
2. Single cell PCR amplification reaction 1
The PCR reaction system was prepared as follows (containing the single cell reverse transcription reaction product):
Figure BDA0003226353900000091
rapid start of single cell PCR amplification reaction 1:
the PCR instrument (Biorad CFX96 PCR) was turned on, the temperature of the hot lid was 105 ℃ and the setting:
1) reacting at 98 ℃ for 3 minutes;
2) (reaction at 98 ℃ for 20 seconds +67 ℃ for 15 seconds +72 ℃ for 6 minutes) × 16 times;
4) reacting at 72 ℃ for 5 minutes;
4) the reaction was terminated at 4 ℃.
3. Purification of Single cell PCR amplification reaction product 1
Note: this step requires the reaction to be carried out in 96-well plates
1) Add 25uL of magnetic beads (V)Magnetic bead/VProduct of1:1), beating for 10 times up and down, fully mixing coffee magnetic beads, and standing for 8 minutes at room temperature.
2) The 96-well plate was placed under magnetic force and allowed to stand for 5 minutes until the solution became clear, and all supernatant was aspirated and discarded (avoiding magnetic beads).
3) The 96-well plate was placed on magnetic plus, 200uL of 80% ethanol was added, left for 30 seconds, aspirated and all supernatant discarded (avoid touching the beads).
4) And repeating the steps.
5) The 96-well plate was placed on a magnetic stand and allowed to air dry at room temperature for 5-10 minutes during which time all residual ethanol was aspirated off using a 10 μ L pipette tip.
6) After the magnetic bead aggregation zone on the tube wall had a fine crack, the 96-well plate was removed from the magnetic stand and 17.5uL of EB (1mM Tris-HCl, or purified water) solution was added. And (4) blowing and beating for 10 times to fully and uniformly mix until the mixture turns brown, and standing for 2 minutes at room temperature.
7) The 96-well plate was placed under magnetic force and allowed to stand for 2 minutes until the solution became clear, and all supernatant was collected (about 17uL, taking care to avoid hitting the beads).
4. Staged quality control 1
1) And (3) carrying out quantitive by a Qubit HS DNA kit on the sample, wherein the normal range of the purified single-cell PCR amplification reaction 1 product is 0.2 ng/uL-2 ng/uL.
2) And carrying out 1uL 2100QC or 8uL electrophoresis QC, wherein the purified single cell PCR amplification reaction 1 product is reasonably distributed into 500-3000bp diffusion bands (representing the average distribution range of the transcript).
5. Tn5 transposase fragmentation reaction
1ng of the purified single-cell PCR amplification reaction 1 product was added to the following reaction system to complete the Tn5 transposase fragmentation reaction.
Figure BDA0003226353900000101
The reaction was left at 55 ℃ for 5 minutes (hot lid closed), after completion of the reaction, the residue on the walls of the centrifuge tubes was centrifuged and placed on ice.
mu.L of NT Buffer was added quickly and reacted at room temperature for 5 minutes, followed by placing on ice.
6. Single cell PCR amplification reaction 2
Figure BDA0003226353900000102
Rapid start of single cell PCR amplification reaction 2:
the PCR instrument (Biorad CFX96 PCR) was turned on, the temperature of the hot lid was 105 ℃ and the setting:
1) reacting for 3 minutes at 72 ℃;
2) reacting at 95 ℃ for 30 seconds;
3)16 cycles x (reaction at 95 ℃ for 10 seconds + reaction at 55 ℃ for 30 seconds + reaction at 72 ℃ for 30 seconds);
4) reacting at 72 ℃ for 5 minutes;
5) the reaction was terminated at 10 ℃.
7. Purification of Single cell PCR amplification reaction product 2
Note: this step requires the reaction to be carried out in 96-well plates
1) 30uL of magnetic beads (V beads/V product: 0.6:1) were added, and the mixture was blown up and down 10 times, and coffee beads were mixed well and left to stand at room temperature for 8 minutes.
2) The 96-well plate was placed under magnetic force and allowed to stand for 5 minutes until the solution became clear, and all supernatant was aspirated and discarded (avoiding magnetic beads).
3) The 96-well plate was placed on magnetic plus, 200uL of 80% ethanol was added, left for 30 seconds, aspirated and all supernatant discarded (avoid touching the beads).
4) And repeating the steps.
5) The 96-well plate was placed on a magnetic stand and allowed to air dry at room temperature for 5-10 minutes, during which time all residual ethanol was aspirated off using a 10uL pipette tip.
6) After the magnetic bead aggregation zone on the tube wall had a fine crack, the 96-well plate was removed from the magnetic stand and 15uL of EB (1mM Tris-HCl, or purified water) solution was added. And (4) blowing and beating for 10 times to fully and uniformly mix until the mixture turns brown, and standing for 2 minutes at room temperature.
7) The 96-well plate was placed under magnetic force and allowed to stand for 2 minutes until the solution became clear, and all supernatant was collected (about 14uL, taking care to avoid hitting the beads).
8. Periodic quality control 2
1) And (3) carrying out quantitive by a Qubit HS DNA kit on the sample, wherein the normal range of the purified single-cell PCR amplification reaction 2 product is 0.5 ng/uL-10 ng/uL.
2) And carrying out 1uL 2100QC or 8uL electrophoresis QC, wherein the purified single cell PCR amplification reaction 2 product is reasonably distributed into 250-1000bp diffusion bands (representing the library length range of adding the adaptor after transposase digestion).
9. Sequencing
1) The samples were diluted to a concentration of 2nM in terms of moles, and mixed at a ratio of 1:1 for each sample. The total amount after mixing was taken to 10uL for sequencing by the sequencing company and 2100QC sequencing was the final.
2) The sequencer used either Hiseq2500 or Hiseq2000, sequencing mode 1x50bp reads long, 250M number of reads.
10. The implementation result of the tumor single cell gene sequencing technology (TuSEQ technology) is as follows:
as shown in fig. 8 and fig. 9, TuSEQ is performed on CTCs separated by TuSTA marker and TuSEP in a patient blood sample by using the method, sequencing analysis identifies specific expression genes and gene mutation results, successfully identifies the nature and tissue source of tumor cells, proves that the tumor cells and the CTCs are respectively from colon cancer patients and pancreatic cancer patients, further provides chemotherapy sensitivity analysis and medication guidance by referring to chemotherapy drug toxic and side gene detection sites according to high-frequency mutation gene information, suggests the use of vinca alkaloids, irinotecan, platinum, pyrimidine analogs, antiestrogens and the like for the colon cancer patients, and suggests the use of podophyllin analogs, pyrimidine analogs, taxanes, antiestrogens and the like for the pancreatic cancer patients. In addition, the personalized tumor vaccine developed aiming at the CTC specific mutant gene realizes the effective killing of tumor cells on a colon cancer mouse model, and as shown in figure 10, the result shows that the tumor volume is obviously reduced after the vaccine immunization in the personalized tumor gene process, and the accurate immunotherapy of the tumor is effectively guided.
In conclusion, the invention has the following beneficial effects:
the embodiment of the invention provides a tumor single cell dual-fluorescence labeling detection Kit, a preparation method and application thereof, wherein a main component of the detection Kit (TuSTA Kit) is deoxyglucose-2- (7-nitrobenzo-2-oxa-1, 3-diazole-4-amino) which is a green fluorescence labeling glucose derivative (FG) and has small molecular weight and is easily and efficiently absorbed by tumor cells, and the other main component Rox-EpCAM-mAb is an EpCAM specific IgG monoclonal antibody molecule marked by a red fluorescence group ROX and is a tumor cell specific labeling molecule, and the detection Kit and the Rox-EpCAM-mAb can indicate the tumor cells when combined for use and have dual specificity; the circulating tumor cells marked by the detection kit can be accurately identified and separated by a tumor single cell counting and separating device system (TuSEP), so that the high-efficiency accurate separation and acquisition of the circulating tumor cells are ensured; the 3TS system composed of the detection kit TuSTA, TuSEP and TuSEQ realizes high-efficiency detection aiming at early circulating tumor cells from stages of identification, separation, acquisition and sequencing analysis, establishes a set of complete tumor precision detection system, and lays a foundation for further carrying out personalized precision treatment on tumor patients.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (10)

1.一种肿瘤单细胞双荧光标记检测试剂盒，其特征在于，包括：试剂A和试剂B，所述试剂A包括由缓冲液配置而成的浓度大于10mg/mL的脱氧葡萄糖-2-(7-硝基苯并-2-氧杂-1，3-二唑-4-氨基)溶液，所述试剂B包括浓度大于1mg/mL的Rox-EpCAM-mAb冻干粉溶液。1. A tumor single cell double fluorescent labeling detection kit is characterized in that, comprising: Reagent A and Reagent B, and described Reagent A comprises deoxyglucose-2-( 7-nitrobenzo-2-oxa-1,3-oxadiazole-4-amino) solution, the reagent B includes a Rox-EpCAM-mAb lyophilized powder solution with a concentration greater than 1 mg/mL. 2.根据权利要求1所述的肿瘤单细胞双荧光标记检测试剂盒，其特征在于，所述试剂A中的缓冲液选用磷酸缓冲液，所述Rox-EpCAM-mAb冻干粉溶液由50％甘油配置而成。2. The tumor single cell double fluorescent labeling detection kit according to claim 1, wherein the buffer in the reagent A is selected from phosphate buffer, and the Rox-EpCAM-mAb lyophilized powder solution is 50% Made with glycerin. 3.根据权利要求2所述的肿瘤单细胞双荧光标记检测试剂盒，其特征在于，所述Rox-EpCAM-mAb冻干粉包括0.01M磷酸缓冲液、1％BSA和0.1％叠氮化钠。3. The tumor single cell double fluorescent labeling detection kit according to claim 2, wherein the Rox-EpCAM-mAb lyophilized powder comprises 0.01M phosphate buffer, 1% BSA and 0.1% sodium azide . 4.一种制备如权利要求1-3中任一项所述的检测试剂盒的方法，其特征在于，包括：4. A method for preparing the detection kit according to any one of claims 1-3, characterized in that, comprising: 称取10mg脱氧葡萄糖-2-(7-硝基苯并-2-氧杂-1，3-二唑-4-氨基)，使用1mL磷酸盐缓冲液溶解，配制成终浓度为10mg/mL的试剂A；Weigh 10 mg of deoxyglucose-2-(7-nitrobenzo-2-oxa-1,3-oxadiazole-4-amino), dissolve it with 1 mL of phosphate buffer, and prepare a final concentration of 10 mg/mL. reagent A; 取1mg Rox-EpCAM-mAb冻干粉溶解于1mL 50％甘油溶液，制成终浓度为1mg/mL的试剂B；Dissolve 1 mg Rox-EpCAM-mAb lyophilized powder in 1 mL of 50% glycerol solution to prepare reagent B with a final concentration of 1 mg/mL; 将试剂A和试剂B于-20℃温度条件下冷藏保存。Store Reagent A and Reagent B refrigerated at -20°C. 5.根据权利要求4所述的方法，其特征在于，所述Rox-EpCAM-mAb冻干粉包括0.01M磷酸缓冲液、1％BSA和0.1％叠氮化钠。5. The method according to claim 4, wherein the Rox-EpCAM-mAb lyophilized powder comprises 0.01M phosphate buffer, 1% BSA and 0.1% sodium azide. 6.权利要求1-3中任一项所述的检测试剂盒在人组织和血液循环肿瘤细胞荧光检测中的应用。6. The application of the detection kit according to any one of claims 1 to 3 in the fluorescence detection of human tissue and blood circulating tumor cells. 7.根据权利要求6所述的应用，其特征在于，包括：7. The application according to claim 6, characterized in that, comprising: 取肿瘤病人或动物的原发灶/转移灶部分组织或抽取3-4mL外周血，肿瘤组织经胰酶消化为单细胞悬液备用，外周血添加抗凝剂备用；Take part of the primary tumor/metastatic tissue of tumor patients or animals or extract 3-4 mL of peripheral blood, the tumor tissue is digested with trypsin to form a single-cell suspension for use, and anticoagulant is added to the peripheral blood for use; 将试剂A以1:100的比例和试剂B以1:1000的比例添加至人肿瘤组织单细胞悬液或外周血样本中后，置含5％二氧化碳、温度为37℃的细胞培养箱中避光培养染色0.5-1h，然后加入3-5倍样本体积的红细胞裂解液，轻轻吹打混匀，室温处理2分钟，取500g离心5min，弃上清，添加1mL磷酸缓冲液重悬细胞，在共聚焦显微镜下检测人肿瘤组织或外周血中细胞荧光染色情况。After adding reagent A at a ratio of 1:100 and reagent B at a ratio of 1:1000 to human tumor tissue single cell suspension or peripheral blood samples, place them in a cell incubator containing 5% carbon dioxide at a temperature of 37 °C to avoid Light culture staining for 0.5-1h, then add 3-5 times the sample volume of red blood cell lysate, gently pipette and mix, treat at room temperature for 2 minutes, centrifuge at 500 g for 5 minutes, discard the supernatant, add 1 mL of phosphate buffer to resuspend the cells, Fluorescence staining of cells in human tumor tissue or peripheral blood was detected under confocal microscope. 8.根据权利要求7所述的应用，其特征在于，还包括将试剂A和试剂B按照10：1配比混合,取25-50μL,微静脉注射荷瘤小鼠,3h后对小鼠进行荧光活体成像观察。8. application according to claim 7, is characterized in that, also comprises that reagent A and reagent B are mixed according to 10: 1 ratio, take 25-50 μ L, venule injects tumor-bearing mice, carries out to mice after 3h. Fluorescence intravital imaging observation. 9.权利要求1-3中任一项所述的检测试剂盒配合肿瘤单细胞基因测序技术鉴定基因突变及表达信息的应用。9. Application of the detection kit according to any one of claims 1 to 3 in combination with tumor single-cell gene sequencing technology to identify gene mutation and expression information. 10.根据权利要求9所述的应用，其特征在于，所述应用基于经检测试剂盒染色标记人组织和血液循环肿瘤细胞后分离出的肿瘤单细胞实施。10 . The application according to claim 9 , wherein the application is implemented based on tumor single cells isolated after staining and labeling human tissue and blood circulating tumor cells with a detection kit. 11 .
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