A test paper and kit for rapid detection of echinocytes

Abstract

The application discloses a test paper and a kit for rapidly detecting echinocytes, and the test paper is composed of a support plate, an anticoagulant sample pad, a connecting pad, a reaction color developing area and an absorption pad; wherein the anticoagulant sample pad and the absorption pad are respectively arranged on the opposite sides of the support plate, the anticoagulant sample pad is connected to one end of the reaction color developing area through the connecting pad, and the other end of the reaction color developing area is connected with the absorption pad. The application is mainly based on a colloidal gold detection method for identifying echinocytes by detecting double-stranded DNA combined on the surface of red blood cells, and the detection method using the test paper and the kit of the application can be realized without specific sites and equipment, is simple, fast and high in accuracy. The test paper and the kit of the application can be used for all clinical cases diagnosed by detecting echinocytes.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological detection, and particularly relates to a test paper and kit for rapidly detecting acanthocytes. BACKGROUND

[0002] Acanthocyte is a red blood cell with needle-like protrusions on the surface, and the distance between the protrusions is irregular. The length and width of the protrusions are not the same. Acanthocytes are generally found in the blood smears of patients with beta lipoprotein deficiency, and can also be found in post-splenectomy and alcoholic liver disease. A research group of Nantong University recently found that different degrees of acanthocyte increase were also found in the blood of a mouse model of highland polycythemia.

[0003] Existing acanthocyte detection technology mainly relies on a microscope to observe and distinguish the morphology, and the application of the detection method is limited by equipment and site. SUMMARY

[0004] In view of the deficiencies of the prior art, the application provides a test paper and kit for rapidly detecting acanthocytes, which reflects the acanthocyte change of red blood cells by measuring the nucleic acid on the surface of the red blood cells.

[0005] The application is implemented by the following technical solutions:

[0006] A test paper for rapidly detecting acanthocytes, the test paper is composed of a support plate, an anticoagulant sample pad, a connecting pad, a reaction color development zone and an absorption pad. The anticoagulant sample pad and the absorption pad are respectively arranged on the support plate and located on opposite sides of the support plate. The anticoagulant sample pad is connected to one end of the reaction color development zone through the connecting pad, and the other end of the reaction color development zone is connected to the absorption pad.

[0007] The anticoagulant sample pad is a Leukosorb cellulose membrane containing EDTA-K2 anticoagulant.

[0008] The connecting pad is a colloidal gold pad, and colloidal gold particles combined with CD47 antibodies are attached to the colloidal gold pad.

[0009] The reaction color development zone includes a nitrocellulose membrane, a detection line and a quality control line. The nitrocellulose membrane is a fluorescent NC membrane. The detection line contains a nucleic acid dye that cannot penetrate the cell membrane of a living cell. The quality control line contains a secondary antibody of CD47.

[0010] Preferably, the pore size of the nitrocellulose membrane is 8 μm.

[0011] Preferably, the nucleic acid dye that cannot penetrate the cell membrane of a living cell is propidium iodide.

[0012] Preferably, the absorption pad is a water-absorbing paper.

[0013] A kit for rapidly detecting spiky red blood cells, comprising the above-mentioned test paper, and a disposable peripheral sampling needle, a sample sampling tube containing 1 mL of an anticoagulant diluent, and a mini portable ultraviolet lamp.

[0014] A kit for diagnosing high altitude polycythemia, comprising the above-mentioned test paper, and a disposable peripheral sampling needle, a sample sampling tube containing 1 mL of an anticoagulant diluent, and a mini portable ultraviolet lamp.

[0015] The beneficial effects of the present application are as follows:

[0016] The present application is mainly based on the colloidal gold detection method for identifying spiky red blood cells by detecting double-stranded DNA combined on the surface of red blood cells. The detection method using the test paper and kit of the present application can be realized without specific site and equipment, is simple and fast, and has high accuracy. The test paper and kit of the present application can be used for all clinical related cases diagnosed by detecting spiky red blood cells. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 It is a scanning electron microscope graph of the red blood cell morphological change in Example 1.

[0018] Figure 2 It is a PCR detection graph of the red blood cell mtDNA content change in Example 2.

[0019] Figure 3 It is a flow cytometry detection graph of the red blood cell surface nucleic acid positive cell group change in Example 3.

[0020] Figure 4 It is a structural schematic diagram of the test paper for rapidly detecting spiky red blood cells in Example 4.

[0021] Figure 4 In the middle: 1, support plate; 2, anticoagulant sample pad; 3, connecting pad; 4, reaction color development area; 5, absorption pad; 6, nitrocellulose membrane; 7, detection line; 8, quality control line.

[0022] Figure 5 It is an operation schematic diagram of the kit for rapidly detecting spiky red blood cells in Example 4.

[0023] Figure 6 It is a detection result judgment schematic diagram of the kit for rapidly detecting spiky red blood cells in Example 4. DETAILED DESCRIPTION

[0024] The present application will be further described in detail below in combination with the drawings and specific embodiments.

[0025] The mouse used in the following examples is C57BL / 6N, which is provided by the animal center of Nantong University.

[0026] Red blood cell spicule changes in mouse blood before and after high altitude exposure

[0027] A low pressure and low oxygen animal device was used to simulate a mouse model exposed to an altitude of 6000 meters. After the mice were continuously exposed for 7 and 14 days, the mouse blood was collected and red blood cell samples were prepared. The morphology of red blood cells in mouse blood was observed by scanning electron microscopy. The specific operation method of red blood cell electron microscopy sample preparation and shooting is as follows:

[0028] After the mice were anesthetized, the blood was collected into an EDTA anticoagulation tube by eyeball blood collection, centrifuged at 500g for 5min, and the plasma and red blood cells were separated. The upper plasma was discarded, and the lower red blood cells were washed with isotonic PBS for three times. Then the sample was fixed with 2.5% (v / v) glutaraldehyde in PBS solution at a volume ratio of 1:10 overnight. After the red blood cells were fixed, the fixed red blood cells were washed with pure water for three times, and then the washed red blood cells were transferred to a silicon wafer and dried overnight at 37°C in a constant temperature condition using a vacuum drying machine. Finally, all samples were coated with a 10nm thick gold film using a sputter coater, and photographed by scanning electron microscopy at 3000 times magnification.

[0029] The results are shown in Figure 1 Compared with the normal pressure and normal oxygen (NN) control group, the red blood cell spicules of the low pressure and low oxygen exposure group for 7 days (HH-7d) and 14 days (HH-14d) were significantly increased, and the 100nM CpG DNA treatment group was the positive control group.

[0030] Example 2: Changes in mtDNA content on the surface of red blood cells in mouse blood before and after high altitude exposure

[0031] In order to verify whether there is mitochondrial DNA (mtDNA) in red blood cells after high altitude exposure, after simulating high altitude 6000 meters for 7 and 14 days, the mouse red blood cells were collected to extract the red blood cell nucleic acid sample, and the mtDNA related genes (ND1, COX1, 16S) were detected by PCR. The specific operation method of red blood cell membrane nucleic acid sample extraction and PCR is as follows:

[0032] After the mice were anesthetized, the blood was collected into an EDTA anticoagulation tube by eyeball blood collection, centrifuged at 500g for 5min, and the plasma and red blood cells were separated. The upper plasma was discarded, and the lower red blood cells were washed with isotonic PBS for three times. The red blood cells were counted using a red blood cell counter. About 1×10 8After the red blood cells were split, the precipitate was collected and 10 μL of YSY Buffer (Nanjing Yaoshunyu Biological Technology Co., Ltd.) was added to lyse the precipitate. The lysis procedure was 65°C for 30 min, 95°C for 5 min, and 16°C for 1 min. 2 μL of the supernatant solution after lysis was added to an eight-tube array, and 10 μL of 2x Rapid Taq Master Mix, 1 μL of the upstream primer of each mitochondrial gene, 1 μL of the downstream primer of each mitochondrial gene, and 6 μL of RNA free water were added to each tube. After the sample was fully suspended, it was placed in a PCR amplifier, and the settings were 95°C for 3 min, 95°C for 15 s, 60°C for 15 s, and 72°C for 15 s, repeated for 35 cycles, and then 72°C for 5 min. After the PCR amplification was completed, 2% agarose gel was used, 10 μL of the sample was subjected to gel electrophoresis, and a photograph was taken for observation.

[0033] The results are shown in Table 1. Figure 2 As shown in Table 1, compared with the normal pressure and normal oxygen (NN) control group, the content of mtDNA in the red blood cells of the mice in the low pressure and low oxygen exposure for 7 days (HH-7d) and 14 days (HH-14d) groups increased significantly, indicating that the increase in mtDNA on the surface of red blood cells caused the increase in the number of spiky red blood cells after high altitude exposure.

[0034] Example 3 In vivo detection of the content of DNA on the surface of red blood cells

[0035] In order to further detect the presence of DNA on the surface of red blood cells in vivo, in this embodiment, after simulating the exposure of mice to high altitude for 7 and 14 days at 6000 meters, the red blood cells of the mice were collected and the samples were incubated with propidium iodide (PI), and flow cytometry was used to analyze the nucleic acid positive cells on the surface of red blood cells. The specific operation method of PI staining on the surface of red blood cells and flow cytometry is as follows:

[0036] After the mice were anesthetized, blood was collected into an EDTA anticoagulant tube by eyeball blood collection, centrifuged at 500 g for 5 min, and the plasma and red blood cells were separated. The upper plasma was discarded, and the lower red blood cells were washed three times with isotonic PBS. 1x10 7 μL of red blood cells were taken, 100 μL of 2% mouse serum was added for blocking for 20 min, 100 μL of PI dye (50 μg / mL, PROTEINBIO, PDA0022) was added at a ratio of 1:20, resuspended, and incubated on ice in the dark for 30 min. After the end, the FL3 (488 nm / 620 nm) channel flow cytometer was used for detection.

[0037] The results are shown in Table 1. Figure 3As shown, compared with the normal pressure and normal oxygen (NN) control group, the proportion of red blood cell surface nucleic acid positive cells in the low pressure and low oxygen exposure 7-day (HH-7d) and 14-day (HH-14d) groups increased significantly, indicating that the content of double-stranded DNA combined on the surface of red blood cells after high altitude exposure increased significantly.

[0038] Example 4

[0039] The experimental results of the above examples 1-3 show that the content of DNA (especially mtDNA) on the surface of red blood cells after high altitude (low pressure and low oxygen) exposure increases significantly, which can further promote the change of red blood cell spiculation. In order to further simplify the method of detecting red blood cell spiculation, this embodiment provides a test paper and kit for rapidly detecting red blood cell spiculation, which is used for rapidly detecting whether a normal person is at risk of suffering from high altitude polycythemia, and the principle is to reflect the change of red blood cell spiculation by measuring the nucleic acid on the surface of red blood cells.

[0040] A test paper for rapidly detecting red blood cell spiculation, as shown in Figure 4 As shown, the test paper is composed of a support plate 1, an anticoagulant sample pad 2, a connecting pad 3, a reaction color development zone 4 and an absorption pad 5, wherein the anticoagulant sample pad 2 and the absorption pad 5 are respectively arranged on the support plate 1 and located on the opposite sides of the support plate 1, the anticoagulant sample pad 2 is connected to one end of the reaction color development zone 4 through the connecting pad 3, and the other end of the reaction color development zone 4 is connected with the absorption pad 5.

[0041] The anticoagulant sample pad 2 is a Leukosorb cellulose membrane containing EDTA-K2 anticoagulant, which can remove white blood cells in whole blood. The connecting pad 3 is a colloidal gold pad, and colloidal gold particles combined with CD47 antibodies are attached to the colloidal gold pad. The absorption pad 5 is a water absorption paper.

[0042] As shown in Figure 4 The reaction color development zone 4 includes a nitrocellulose membrane 6, a detection line 7 and a quality control line 8; the nitrocellulose membrane 6 is a fluorescent NC membrane with a pore size of 8 μm; the detection line 7 contains a nucleic acid dye that cannot penetrate the cell membrane of living cells, such as propidium iodide; and the quality control line 8 contains a secondary antibody of CD47.

[0043] Principle of detection: when the red blood cell surface binds to DNA containing CpG sequence, it will cause the acanthosis of red blood cell, at this time the acanthosis red blood cell surface can be combined with nucleic acid dye (detection line 7), under ultraviolet irradiation, it shows nucleic acid positive; while the CD47 on the surface of acanthosis red blood cell cannot be detected by CD47 antibody (quality control line 8) due to conformational change, it shows negative. At the same time, since the normal red blood cell surface does not contain DNA containing CpG sequence, the nucleic acid dye (detection line 7) cannot detect the nucleic acid on the surface of red blood cell after incubation, which shows negative; while the CD47 on the surface of normal red blood cell is normal, so it can be detected by CD47 antibody (quality control line 8), which shows positive. In summary, whether there is acanthosis red blood cell in human body or not, normal red blood cell must account for the majority compared with acanthosis red blood cell, so the quality control line 8 must develop color, and only in the case of acanthosis red blood cell, the detection line 7 will develop color, and the normal red blood cell will not make the detection line 7 develop color. Based on this, it can be measured whether there is acanthosis red blood cell in blood, and further judge whether normal people have the risk of suffering from high altitude polycythemia.

[0044] A kit for rapid detection of acanthosis red blood cell, comprising the above-mentioned detection test paper (packed in an aluminum foil bag), and a disposable peripheral sampling needle, a sample sampling tube containing 1 mL of anticoagulant diluent, a mini portable ultraviolet lamp and an instruction manual.

[0045] The operation method of the above-mentioned kit for rapid detection of acanthosis red blood cell is as shown in Figure 5 , and the specific steps are as follows:

[0046] (1) Blood sample acquisition: take a disposable peripheral sampling needle to take 1 drop of blood at the ring finger and drop it into the sample sampling tube and mix well.

[0047] (2) Take out the detection test paper in the aluminum foil bag, drop 5 drops of diluted sample liquid at the anticoagulant sample pad, and place it flat for 15 min.

[0048] (3) Open the mini portable ultraviolet lamp to irradiate the reaction color developing area, and read the detection result according to the instruction manual, as shown in Figure 6 , wherein the quality control line (C) does not develop color, which is invalid, when the quality control line (C) develops color, the detection line (T) develops color, which is positive, and does not develop color, which is negative.

[0049] The above is only part of the preferred embodiments of the present application, neither the text nor the drawings can limit the protection scope of the present application, any equivalent structural transformation made by using the content of the present application specification and drawings, or direct / indirect application in other related technical fields shall be included in the protection scope of the present application.

Claims

1. A test strip for rapid detection of echinocytes, characterized in that, The detection test paper is composed of a support plate, an anticoagulant sample pad, a connecting pad, a reaction color developing area and an absorbing pad; wherein the anticoagulant sample pad and the absorbing pad are respectively arranged on the support plate and located on opposite sides of the support plate, the anticoagulant sample pad is connected to one end of the reaction color developing area through the connecting pad, and the other end of the reaction color developing area is connected with the absorbing pad; the anticoagulant sample pad is a Leukosorb cellulose membrane containing EDTA-K2 anticoagulant; The connecting pad is a colloidal gold pad, and colloidal gold particles combined with CD47 antibodies are attached to the colloidal gold pad; the reaction color developing area comprises a nitrocellulose membrane, a detection line and a quality control line; the nitrocellulose membrane is a fluorescent NC membrane; the detection line contains a nucleic acid dye that cannot penetrate the cell membrane of a living cell; and the quality control line contains a secondary antibody of CD47.

2. The test paper for rapidly detecting echinocytes according to claim 1, characterized by The pore size of the nitrocellulose membrane is 8 μm.

3. The test paper for rapidly detecting echinocytes according to claim 1, characterized by The nucleic acid dye that cannot penetrate the cell membrane of a living cell is propidium iodide.

4. The test paper for rapidly detecting echinocytes according to claim 1, wherein The absorbing pad is water-absorbing paper.

5. A kit for rapid detection of echinocytes, characterized by, The kit comprises the detection test paper according to any one of claims 1-4, a disposable peripheral sampling needle, a sample sampling tube containing 1 mL of anticoagulant diluent and a mini portable ultraviolet lamp.

6. A kit for diagnosing high altitude polycythemia, characterized by, The kit comprises the detection test paper according to any one of claims 1-4, a disposable peripheral sampling needle, a sample sampling tube containing 1 mL of anticoagulant diluent and a mini portable ultraviolet lamp.

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