Detection kit for simultaneously identifying virus infection, bacterial infection and immune injury and use method thereof

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to a detection kit for simultaneously identifying virus infection, bacterial infection and immune injury and a use method of the detection kit. According to the scheme, the specific molecular expression level of leucocyte subgroups of pathogenic bacterium infected patients is detected, and a CD64 index, a CD169 index and an HLA-DR index are established as evaluation indexes; virus and bacterium infection and the state of the immune function in a patient after infection can be distinguished at the same time, auxiliary parameters for evaluating the treatment effect are provided for clinicians, and the detection efficiency and the diagnosis and treatment speed are greatly improved.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and specifically relates to a detection kit for simultaneously identifying viral infection, bacterial infection and immune damage and a method for using the same. Background Art

[0002] Infection is one of the common clinical diseases and one of the important causes of death in critically ill patients. Infection occurs in any part of the human body. The presence of infection indicates that the body has been invaded by a certain pathogen, which may lead to tissue damage, inflammatory response and a series of clinical symptoms. Therefore, the diagnosis of infection is crucial for determining treatment plans and prognosis assessment. Different types of infections require different treatment plans. For example, bacterial infections usually require antibiotics for treatment, while viral infections may require antiviral drugs or supportive treatment. Through a comprehensive analysis of the clinical manifestations of infection, laboratory test results and pathogen detection results, doctors can develop targeted treatment plans to improve treatment outcomes and reduce the occurrence of complications.

[0003] In the prior art, a Chinese patent with publication number CN113945712A provides a detection method and a detection kit for distinguishing bacterial and viral infections. By jointly detecting three markers, TRAIL, IP-10, and CRP, when two or more markers are positive, it is judged as viral infection, and when two or more markers are negative, it is judged as bacterial infection, which can significantly improve the accuracy of detection. However, the above prior art can only judge bacterial infection or viral infection by detecting markers, and cannot judge the degree of infection of patients and detect the two indicators simultaneously.

[0004] There are also kits for single detection of bacterial infection, viral infection or immune damage in the prior art, but patients with complex conditions need multiple sampling tests, which is time-consuming and labor-intensive, and has low detection efficiency. Therefore, it is particularly important to develop a technical solution that can quickly detect and diagnose the type of infection, and can accurately reflect bacterial infection, viral infection and immune damage from laboratory indicators, so as to provide a reference for treatment. Summary of the invention

[0005] In view of the problem that the existing detection kits mentioned in the background technology can only detect a single infection index or immune status of the body, resulting in low detection efficiency, the present invention provides a detection kit and a method of using the kit for simultaneously identifying viral infection, bacterial infection and immune damage. The method detects the expression level of specific molecules of leukocyte subgroups in patients infected with pathogenic bacteria, and can simultaneously distinguish between viral and bacterial infections and the state of immune function in patients after infection, providing clinicians with auxiliary parameters for evaluating the treatment effect, greatly improving the detection efficiency and diagnosis and treatment speed. The specific technical scheme is as follows:

[0006] First, the present invention provides a detection kit for simultaneously identifying viral infection, bacterial infection and immune damage, the detection kit comprising a phenotypic detection fluorescently labeled antibody, a hemolytic agent, an experimental buffer and a washing buffer, the phenotypic detection fluorescently labeled antibody comprising a CD11b fluorescently labeled antibody, a CD14 fluorescently labeled antibody, a CD45 fluorescently labeled antibody, a CD64 fluorescently labeled antibody, a CD169 fluorescently labeled antibody and an HLA-DR fluorescently labeled antibody.

[0007] Furthermore, the fluorescent label includes any one of FITC, PE, PE-Cy7, PerCP, APC and APC-Cy7.

[0008] Furthermore, the hemolytic agent includes diglycosides, formaldehyde, and a buffer solution.

[0009] Furthermore, the experimental buffer is a phosphate buffer containing 1% BSA; and the washing buffer is a phosphate buffer containing tween-20.

[0010] The present invention also provides a method for using a detection kit for simultaneously identifying viral infection, bacterial infection and immune damage, comprising the following steps:

[0011] Step 1: Add blood sample, fluorescent labeled antibody for phenotypic detection and experimental buffer to the flow cytometer tube, and incubate at room temperature in the dark with shaking;

[0012] Step 2: After the incubation is completed, add a hemolytic agent to the flow cytometer tube, mix well, keep it in a dark place, and then centrifuge to remove the supernatant; then add a washing buffer to the flow cytometer tube, vortex and centrifuge to remove the supernatant, then add a washing buffer to the fixed volume, vortex and resuspend, and place it in a flow cytometer to obtain a flow cytometric analysis scatter plot;

[0013] Step 3: Based on the FSC / SSC characteristics, the active leukocyte subpopulations were circled in the flow cytometry analysis scatter plot, and the leukocyte subpopulations were preliminarily distinguished by CD45 expression within the leukocyte subpopulations. The leukocyte subpopulations were analyzed based on the dual parameters of CD11b and CD14 expression, and the leukocyte subpopulations were divided into neutrophils, monocytes and lymphocytes;

[0014] Step 4. Calculate the CD64 index, CD169 index and HLA-DR index according to the average fluorescence intensity of CD64, CD169 and HLA-DR in neutrophils, monocytes and lymphocytes, and judge the expression of CD64, CD169 and HLA-DR according to the CD64 index, CD169 index and HLA-DR index, and then judge the infection and immune damage of the sample.

[0015] Furthermore, the calculation formula of the CD64 index is: CD64 index = average value of neutrophil CD64 fluorescence intensity / average value of lymphocyte CD64 fluorescence intensity;

[0016] The calculation formula of CD169 index is: CD169 index = mean fluorescence intensity of monocyte CD169 / mean fluorescence intensity of lymphocyte CD169;

[0017] The calculation formula of HLA-DR index is: mean fluorescence intensity of monocyte HLA-DR / mean fluorescence intensity of neutrophil HLA-DR.

[0018] Furthermore, in step three, cells that are CD11b positive and CD14 negative are neutrophils, cells that are CD11b positive and CD14 positive are monocytes, and cells that are CD11b negative and CD14 negative are lymphocytes.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1) There are many kits for detecting single indicators in the existing market, such as detecting the expression levels of CD64 and HLA-DR molecules, so as to evaluate the bacterial infection of samples and the immune function of patients. When the sample is subjected to multi-indicator detection and evaluation, it is necessary to use multiple single-indicator detection kits to complete the detection and evaluation tasks, which will undoubtedly affect the efficiency of diagnosis and treatment. The detection kit provided by the present invention screens antigen indicators that can accurately reflect the bacterial infection, viral infection and immune function of the sample, and combines three types of detection antibodies in the same detection kit. Through flow cytometry and fluorescence intensity analysis, it is possible to simultaneously accurately evaluate the bacterial infection, viral infection and immune function of the sample, realize the integration of diagnosis, typing, treatment detection and prognosis evaluation, and is more suitable for the detection of complex samples, greatly improving the detection efficiency. At the same time, the present invention simultaneously detects multiple detection indicators, which will not interfere with each other and can corroborate each other, and can provide more comprehensive reference information for the diagnosis, treatment and prognosis evaluation of patients, avoiding the problem of interference of non-infectious factors in the detection process of the single indicator kit.

[0021] 2) The kit provided by the present invention is based on flow cytometry. The leukocyte subpopulation that can accurately express the target antibody is first selected by CD45 expression, and the leukocyte subpopulation is analyzed by the dual parameters of CD11b and CD14 expression, and the leukocyte subpopulation is divided into neutrophils, monocytes and lymphocytes. Finally, the CD64 index, CD169 index and HLA-DR index are calculated by counting the average fluorescence intensity of the specific expression antibody in the corresponding cells, thus avoiding the interference of non-infectious factors in the sample, and accurately reflecting the infection and immune performance of the sample, thereby improving the accuracy of the detection. At the same time, by establishing the CD64 index, CD169 index and HLA-DR index as evaluation indicators, the user can establish an evaluation database through the accumulation and analysis of sample data during use, and realize more accurate stratified diagnosis for samples of various conditions, which has broad prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The flow cytometry analysis scatter plot in Example 1 of the present invention;

[0023] Figure 2 This is a diagram of subcellular population analysis in Example 1 of the present invention;

[0024] Figure 3 This is a comparison chart of the mean fluorescence intensity of antibodies of the test sample and the normal sample in Example 1 of the present invention;

[0025] Figure 4 This is a comparison chart of the mean fluorescence intensity of antibodies of the test sample and the normal sample in Example 2 of the present invention. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the present invention more clear, the technical solution of the present invention will be clearly and completely described below in conjunction with embodiments and drawings.

[0027] The present invention provides a detection kit for simultaneously identifying viral infection, bacterial infection and immune injury, including phenotypic detection fluorescently labeled antibodies, hemolytic agents, experimental buffers and washing buffers, wherein the phenotypic detection fluorescently labeled antibodies include CD11b fluorescently labeled antibodies, CD14 fluorescently labeled antibodies, CD45 fluorescently labeled antibodies, CD64 fluorescently labeled antibodies, CD169 fluorescently labeled antibodies and HLA-DR fluorescently labeled antibodies. Fluorescent labels include but are not limited to any one of FITC, PE, PE-Cy7, PerCP, APC and APC-Cy7. Specifically, in the present invention, CD11b is fluorescently labeled as PE-Cy7; CD14 is fluorescently labeled as FITC; CD45 is fluorescently labeled as PerCP; CD64 is fluorescently labeled as PE; CD169 is fluorescently labeled as APC-Cy7; and HLA-DR is fluorescently labeled as APC.

[0028] The hemolytic agent of the present invention is prepared from diglycosides, formaldehyde and buffer solution. The experimental buffer solution is a phosphate buffer solution containing 1% BSA, and the washing buffer solution is a phosphate buffer solution containing tween-20.

[0029] The present invention also provides a method for using a detection kit for simultaneously identifying viral infection, bacterial infection and immune damage, comprising the following steps:

[0030] Step 1: Add blood sample, fluorescent labeled antibody for phenotypic detection and experimental buffer to the flow cytometer tube, and incubate at room temperature in the dark with shaking;

[0031] Specifically, collect the anticoagulated blood sample of the sample to be tested, add 100 μl of the sample, 1 to 10 μl of phenotypic detection fluorescent labeled antibody and 2 ml of experimental buffer to the flow cytometer, the phenotypic detection fluorescent labeled antibody is as described above, including PE-Cy7 labeled CD11b antibody, FITC labeled CD14 antibody, PerCP labeled CD45 antibody, PE labeled CD64 antibody, APC-Cy7 labeled CD169 antibody and APC labeled HLA-DR antibody. Incubate the flow cytometer for 30-60 minutes at room temperature with shaking in the dark.

[0032] Step 2: After the incubation is completed, add a hemolytic agent to the flow cytometer tube, mix well, keep it in a dark place, and then centrifuge to remove the supernatant; then add a washing buffer to the flow cytometer tube, vortex and centrifuge to remove the supernatant, then add a washing buffer to the fixed volume, vortex and resuspend, and place it in a flow cytometer to obtain a flow cytometric analysis scatter plot;

[0033] Specifically, after the incubation is completed, add 2 ml of hemolytic agent to the flow cytometer tube, mix on a vortex mixer for 10 seconds, and then stand at room temperature in the dark for 10 minutes to lyse the red blood cells. After the lysis is completed, centrifuge at 1000-1500 rpm for 5 minutes, discard the supernatant, add 2 ml of washing buffer, vortex for 3-10 seconds, and centrifuge at 1000-1500 rpm for 5 minutes. Discard the supernatant, add 100 μl of washing buffer to each tube according to the flow cytometer loading requirements, vortex for 8-12 seconds to resuspend, and load the flow cytometer for detection.

[0034] Step 3: Based on the FSC / SSC characteristics, the active leukocyte subpopulations were circled in the flow cytometry analysis scatter plot, and the leukocyte subpopulations were preliminarily distinguished by CD45 expression within the leukocyte subpopulations. The leukocyte subpopulations were analyzed based on the dual parameters of CD11b and CD14 expression, and were distinguished into neutrophils, monocytes and lymphocytes;

[0035] Specifically, according to the flow cytometry analysis scatter plot, dead cells, debris and adhesions are excluded based on the FSC / SSC characteristics, and the active leukocyte subpopulations are circled. The leukocyte subpopulations are initially distinguished by CD45 expression within the leukocyte subpopulations, and CD45-positive cells are circled for dual-parameter analysis of CD11b and CD14 expression. CD11b-positive CD14-negative cells are judged to be neutrophils, CD11b-positive CD14-positive cells are monocytes, and CD11b-negative CD14-negative cells are lymphocytes.

[0036] Step 4. Calculate the CD64 index, CD169 index and HLA-DR index according to the average fluorescence intensity of CD64, CD169 and HLA-DR in neutrophils, monocytes and lymphocytes, and judge the expression of CD64, CD169 and HLA-DR according to the CD64 index, CD169 index and HLA-DR index, and then judge the infection and immune damage of the sample.

[0037] Specifically, the average fluorescence intensity of CD64, CD169 and HLA-DR is analyzed according to the differentiated neutrophils, monocytes and lymphocytes, and the CD64 index, CD169 index and HLA-DR index are calculated according to the following calculation formulas. CD64 index = average fluorescence intensity of neutrophil CD64 / average fluorescence intensity of lymphocyte CD64; the calculation formula of CD169 index is: average fluorescence intensity of monocyte CD169 / average fluorescence intensity of lymphocyte CD169; the calculation formula of HLA-DR index is: average fluorescence intensity of monocyte HLA-DR / average fluorescence intensity of neutrophil HLA-DR.

[0038] The expression of CD64 in neutrophils, CD169 in monocytes and HLA-DR in monocytes is determined based on the CD64 index, CD169 index and HLA-DR index, thereby determining the infection and immune damage of the sample.

[0039] Example 1

[0040] In this embodiment, a detection kit for simultaneously identifying viral infection, bacterial infection and immune damage is as described above. The sample in the method of use is from an actual test sample, steps one and two are also as described above, and steps three and four are as shown in the attached Figure 1-3 shown.

[0041] Attached Figure 1 This is a scatter plot of flow cytometry analysis in this example. Based on the FSC / SSC characteristics, dead cells, debris and adhesions were excluded and the viable leukocyte subpopulation (in the black box) was circled.

[0042] Attached Figure 2The left picture shows the preliminary differentiation of leukocyte subsets by CD45 expression, and the cells with positive CD45 expression are circled. The right picture shows the dual parameter analysis of CD11b and CD14 expression to differentiate neutrophils, monocytes and lymphocytes.

[0043] Attached Figure 3 From left to right, the mean fluorescence intensity of neutrophils, monocytes and mononuclear cells expressing CD64, HLA-DR and CD169 (upper figure) and the comparison of the mean fluorescence intensity of normal expression of CD64, HLA-DR and CD169 (lower figure) are shown.

[0044] Analysis of test results: The expression of CD64 on neutrophils in the test samples was significantly increased compared with the expression of CD64 on lymphocytes in normal samples. The CD64 index was calculated to be 15.7, which was interpreted as moderate upregulation of CD64 expression.

[0045] The single-cell HLA-DR expression in the test sample did not change significantly compared with the single-cell HLA-DR expression in the normal sample, which was interpreted as normal HLA-DR expression.

[0046] The expression of CD169 on single cells in the test sample was not significantly different from that on lymphocytes in normal samples, and the calculated CD169 index was 5.72, which was interpreted as normal CD169 expression. The final result showed that the sample was accompanied by bacterial infection and had normal immune function.

[0047] Example 2

[0048] The samples in this embodiment are from a certain actual test sample. The other experimental steps are consistent with the embodiment, and the test results are shown in the attached Figure 4 shown.

[0049] Analysis of test results: The expression of CD64 on neutrophils in the test samples was significantly increased compared with the expression of CD64 on lymphocytes in normal samples. The CD64 index was calculated to be 11.5, which was interpreted as moderate upregulation of CD64 expression.

[0050] The single-cell HLA-DR expression in the test samples was significantly decreased compared with the single-cell HLA-DR expression in the normal samples, which was interpreted as severe downregulation of HLA-DR expression.

[0051] The expression of CD169 in single cells in the test sample was slightly increased compared with the expression of CD169 in lymphocytes in normal samples, and the calculated CD169 index was 6.58, which was interpreted as a slight increase in CD169 expression. The results indicated that the patient was infected with bacteria and viruses at the same time, and the body's immune function was severely damaged.

[0052] It is obvious to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be regarded as exemplary and non-restrictive from any point of view. In addition, it should be understood that although this specification is described in accordance with the embodiments, it does not contain only one technical solution. This narrative of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in the embodiments can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A detection kit for simultaneously identifying viral infection, bacterial infection and immune damage, characterized in that: The detection kit comprises a phenotype detection fluorescently labeled antibody, a hemolytic agent, an experimental buffer and a washing buffer, and the phenotype detection fluorescently labeled antibody comprises a CD11b fluorescently labeled antibody, a CD14 fluorescently labeled antibody, a CD45 fluorescently labeled antibody, a CD64 fluorescently labeled antibody, a CD169 fluorescently labeled antibody and an HLA-DR fluorescently labeled antibody.

2. A detection kit for simultaneously identifying viral infection, bacterial infection and immune damage according to claim 1, characterized in that: The fluorescent label includes any one of FITC, PE, PE-Cy7, PerCP, APC and APC-Cy7.

3. A detection kit for simultaneously identifying viral infection, bacterial infection and immune damage according to claim 2, characterized in that: The hemolytic agent includes diglycosides, formaldehyde and a buffer.

4. A detection kit for simultaneously identifying viral infection, bacterial infection and immune damage according to claim 2, characterized in that: The experimental buffer is a phosphate buffer containing 1% BSA; the washing buffer contains a phosphate buffer containing tween-20.

5. A method for using a detection kit for simultaneously identifying viral infection, bacterial infection and immune damage as described in any one of claims 1 to 4, characterized in that: The following steps are involved: Step 1: Add blood sample, fluorescent labeled antibody for phenotypic detection and experimental buffer to the flow cytometer tube, and incubate at room temperature in the dark with shaking; Step 2: After the incubation is completed, add a hemolytic agent to the flow cytometer tube, mix well, keep it in a dark place, and then centrifuge to remove the supernatant; then add a washing buffer to the flow cytometer tube, vortex and centrifuge to remove the supernatant, then add a washing buffer to the fixed volume, vortex and resuspend, and place it in a flow cytometer to obtain a flow cytometric analysis scatter plot; Step 3: Based on the FSC / SSC characteristics, the active leukocyte subpopulations were circled in the flow cytometry analysis scatter plot, and the leukocyte subpopulations were preliminarily distinguished by CD45 expression within the leukocyte subpopulations. The leukocyte subpopulations were analyzed based on the dual parameters of CD11b and CD14 expression, and the leukocyte subpopulations were divided into neutrophils, monocytes and lymphocytes; Step 4. Calculate the CD64 index, CD169 index and HLA-DR index according to the average fluorescence intensity of CD64, CD169 and HLA-DR in neutrophils, monocytes and lymphocytes, and judge the expression of CD64, CD169 and HLA-DR according to the CD64 index, CD169 index and HLA-DR index, and then judge the infection and immune damage of the sample.

6. A method for using a detection kit for simultaneously identifying viral infection, bacterial infection and immune damage according to claim 5, characterized in that: In the step 1, the volume ratio of the blood sample to the phenotype detection fluorescent labeled antibody is 100:1-10; the shaking incubation time is 30-60 minutes.

7. A method for using a detection kit for simultaneously identifying viral infection, bacterial infection and immune damage according to claim 5, characterized in that: In the step 2, the light-proof standing time is 10 to 15 minutes; the centrifugal speed is 1000 to 1500 r / min, and the centrifugal time is 5 to 10 minutes.

8. A method for using a detection kit for simultaneously identifying viral infection, bacterial infection and immune damage according to claim 5, characterized in that: In the step 3, cells that are CD11b positive and CD14 negative are neutrophils, cells that are CD11b positive and CD14 positive are monocytes, and cells that are CD11b negative and CD14 negative are lymphocytes.

9. A method for using a detection kit for simultaneously identifying viral infection, bacterial infection and immune damage according to claim 5, characterized in that: The calculation formula of the CD64 index in step 4 is: CD64 index = average value of neutrophil CD64 fluorescence intensity / average value of lymphocyte CD64 fluorescence intensity; The calculation formula of CD169 index is: CD169 index = mean fluorescence intensity of monocyte CD169 / mean fluorescence intensity of lymphocyte CD169; The calculation formula of HLA-DR index is: HLA-DR index = monocyte HLA-DR mean fluorescence intensity / neutrophil HLA-DR mean fluorescence intensity.

Images