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Method for differential diagnosis of EBV infected cell subtype and application thereof

A technology for infecting cells and differential diagnosis, applied in the medical field, can solve the problems of inconvenient tissue biopsy, high instrument requirements, poor sensitivity, etc., to ensure high sensitivity, high specificity, high clinical application value, reliable detection and identification. Effect

Pending Publication Date: 2022-03-22
CHILDRENS HOSPITAL OF CHONGQING MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because in the initial stage of the disease caused by EBV infection, the body has increased EBV viral load and lymphoid cell proliferation, conventional PCR detection and hematological antibody detection can only determine the total EBV-DNA load in the patient, and it is difficult to distinguish the subtype of EBV-infected cells. type
At present, the identification of EBV-infected cell subpopulations commonly used in clinical practice is based on PCR detection or tissue biopsy based on cell sorting technology. Infected cells or plasma EBV-DNA contamination of uninfected cells lead to false positive results, poor sensitivity; tissue biopsy is often inconvenient to obtain materials, which is difficult to meet the needs of clinical testing

Method used

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  • Method for differential diagnosis of EBV infected cell subtype and application thereof
  • Method for differential diagnosis of EBV infected cell subtype and application thereof
  • Method for differential diagnosis of EBV infected cell subtype and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0057] Application of Flow-FISH technology in cell lines and clinical patients

[0058] In this example, flow cytometry and fluorescence in situ hybridization are combined, and EBERs is used as the target RNA to hybridize with EBERs probes, and the expression of EBERs and surface antigens is detected simultaneously to identify the cell subtype infected with the virus. The specific experimental process is as follows:

[0059] 1. Research object

[0060] The research objects of this embodiment are EBV-negative and EBV-positive human cell lines cultured in vitro, and peripheral blood samples of clinical patients.

[0061] (1) EBV negative (EBV-) cell lines: human B lymphocytic leukemia cell line Sup-B15, human acute T cell leukemia cell line Molt-4.

[0062] EBV positive (EBV+) cell lines: marmoset EB virus transformed leukocyte line B95-8, human Burkitt's tumor cell line Raji.

[0063] The characteristics and sources of the above cell lines are shown in Table 2.

[0064] Tab...

Embodiment 2

[0108] Under normal circumstances, fluorescently labeled probes cannot enter the cell through the intact cell membrane. To measure RNA in the nucleus by flow cytometry, it is necessary to use a membrane-breaking agent to destroy the integrity of the cell membrane to create small holes for the probe to pass through. The effect of membrane disruption will directly affect the fluorescence staining and result analysis.

[0109] Based on this, referring to the experimental method of Example 1, this example explores the type of immobilized membrane-breaking agent suitable for flow cytometry-fluorescence in situ hybridization. The experimental method and process are as follows:

[0110] 1. By different membrane breaking agents: 0.2% TritonX-100, 0.2% Saponin, 0.2% Tween-20, and Thermo Fisher commercially available finished membrane breaking kits (TritonX-100, Saponin are all purchased from sigma-aldrich Sigma Aldrich (Shanghai) Trading Co., Ltd.; Tween-20 was purchased from Beijing S...

Embodiment 3

[0124] At present, there is no kit specifically for flow cytometry-fluorescence in situ hybridization on the market, all of which contain hybridization solutions (including 30% formamide and 10% dextran sulfate) designed for traditional fluorescence in situ hybridization. The state requirements are not high. In order to increase the fluorescence intensity of the probe, a high-concentration hybridization solution is usually used, but this has a great impact on the cell state, and it cannot be detected by flow cytometry when it is applied in the method of the present invention. Therefore, in this embodiment, the concentration of the main components (formamide and dextran sulfate) in the hybridization solution containing the probe was explored to find out the concentration of the hybridization solution suitable for flow cytometry-fluorescence in situ hybridization.

[0125] The main components of the hybridization solution used in this example are formamide and dextran sulfate. A...

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Abstract

The invention belongs to the field of medical science, and particularly discloses a method for differential diagnosis of EBV infected cell subtypes and application thereof, and the method comprises the following steps: staining cells by using a fluorescence labeled lymphocyte specific antibody, specifically recognizing EBV infection characteristic RNA: EBERs in the cells by using a FISH probe, and then detecting EBV infected cell subgroups by flow cytometry, the cell subtype infected by the EBV is identified. The flow cytometry and the in-situ fluorescence hybridization are combined for use, a method capable of directly, quickly, conveniently and reliably detecting and identifying the EBV infected cell subtype of lymphocytes in a clinical peripheral blood sample is established, and the method has extremely high clinical application value.

Description

technical field [0001] The invention relates to the field of medical technology, in particular to a method for differentially diagnosing EBV-infected cell subtypes and its application. Background technique [0002] EBV (Epstein-Barr Virus, EBV) virus is a type 4 herpes virus, and it is also the first virus found to be associated with tumors. The virus has an infection rate of 90% in the population, but most of them are asymptomatic infections. The main target cells of EBV in the human body are B cells and oral epithelial cells. In addition, a small amount of T cells and NK cells can also infect EBV. Usually, EBV enters B cells through CD21 and then replicates and proliferates to produce surface antigens. This process is recognized by the body's immune system to generate specific CD8+ T cells to kill actively infected B cells and control virus expansion. A small number of B cells carrying quiescent EBV infection are not recognized and killed by T cells, forming a latent inf...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6841G01N33/569G01N33/58C12R1/93
CPCC12Q1/705C12Q1/6841G01N33/56966G01N33/582G01N2469/10C12Q2563/107C12Q2527/125
Inventor 舒逸苏虹宇邹琳傅国何晓燕
Owner CHILDRENS HOSPITAL OF CHONGQING MEDICAL UNIV
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