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Method for detecting virulence of legionella pneumophila

A technology of Legionella pneumophila and virulence, applied in the field of detection of Legionella pneumophila virulence, can solve the problems of poor repeatability, cumbersome operation, insufficient sensitivity, etc., and achieve intuitive and accurate virulence strength, great clinical significance and the effect of the reference value

Inactive Publication Date: 2020-08-18
南京实践医学检验有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The traditional solid-phase biochip (Biochip) or gene chip technology has outstanding weaknesses such as poor repeatability, insufficient sensitivity, and cumbersome operation. Therefore, we need a new method for detecting the virulence of Legionella pneumophila to solve the above problems. satisfy people's demands

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0021] A method for detecting the virulence of Legionella pneumophila comprises the following steps in sequence:

[0022] Step 1: Cultivate the strain of Legionella pneumophila to be tested. To cultivate the strain of Legionella pneumophila to be tested, the strain of Legionella pneumophila to be tested is streaked and inoculated onto a BCYE solid medium plate, and placed at 37°C, 5% CO 2 Cultivate in the environment for 48 hours. The preparation method of BCYE solid medium is to dissolve 12.5g of carbon powder in ddH2O to a volume of 450ml, and autoclave at 121°C for 30min; Dissolve the Legionella Supplement and mix the two under aseptic conditions:

[0023] Step 2: Culture HEK293T / 17 cells. To culture HEK293T cells, take a 6-well plate, spread 5x104 HEK293T / 17 cells in each well, and place at 37°C, 5% CO 2 cultivated in the environment;

[0024] Step 3: Construct the target gene into the vector: DNA extraction is performed on Legionella pneumophila, then construct the vect...

Embodiment 2

[0029] A method for detecting the virulence of Legionella pneumophila comprises the following steps in sequence:

[0030] Step 1: Cultivate the strain of Legionella pneumophila to be tested. To cultivate the strain of Legionella pneumophila to be tested, the strain of Legionella pneumophila to be tested is streaked and inoculated onto a BCYE solid medium plate, and placed at 37°C, 5% CO 2 Cultivate in the environment for 48 hours. The preparation method of BCYE solid medium is to dissolve 12.5g of carbon powder in ddH2O to a volume of 450ml, and autoclave at 121°C for 30min; Dissolve the Legionella Supplement and mix the two under aseptic conditions:

[0031] Step 2: Culture HEK293T / 17 cells. To culture HEK293T cells, take a 6-well plate, spread 5x104 HEK293T / 17 cells in each well, and place at 37°C, 5% CO 2 cultivated in the environment;

[0032] Step 3: Construct the target gene into the vector: DNA extraction is performed on Legionella pneumophila, then construct the vect...

Embodiment 3

[0037] A method for detecting the virulence of Legionella pneumophila comprises the following steps in sequence:

[0038] Step 1: Cultivate the strain of Legionella pneumophila to be tested. To cultivate the strain of Legionella pneumophila to be tested, the strain of Legionella pneumophila to be tested is streaked and inoculated onto a BCYE solid medium plate, and cultured in an environment of 37°C and 5% CO2 For 48 hours, the preparation method of BCYE solid medium is to dissolve 12.5g of carbon powder in ddH2O to a volume of 450ml, and autoclave at 121°C for 30min; when the temperature is lowered to 50°C, dissolve Legionella in 50ml of sterilized ddH2O Additives, mix the two under sterile conditions:

[0039] Step 2: Culture HEK293T / 17 cells. To culture HEK293T cells, take a 6-well plate, spread 5x104 HEK293T / 17 cells in each well, and place at 37°C, 5% CO 2 cultivated in the environment;

[0040] Step 3: Construct the target gene into the vector: DNA extraction is perfor...

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Abstract

The invention discloses a method for detecting the virulence of legionella pneumophila. The method sequentially comprises the following steps: 1, culturing a legionella pneumophila strain to be detected; 2, culturing HEK293T / 17 cells; 3, constructing a target gene to a vector; 4, transfecting HEK293T / 17 cells: paving a plate and infecting the HEK293T / 17 cells; 5, transfection detection: turning ona fluorescence device, placing a cell culture plate on an objective table, and observing whether fluorescence exists or not through a fluorescence channel to confirm that infection succeeds or fails;and 6, culturing the successfully transfected HEK293T / 17 cells, infecting mice in a nasal dripping manner, and judging the virulence of the legionella pneumophila to be detected by observing the death rate the lung tissue damage degree of the mice and cytokine detection. The method for detecting the virulence of legionella pneumophila can reflect the real virulence of legionella pneumophila in ahost body more accurately, is more intuitive and accurate, has greater clinical significance and reference value, and has positive influence on prevention and control of legionella pneumophila.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a method for detecting the virulence of Legionella pneumophila. Background technique [0002] Legionella, an aerobic Gram-negative coccus, is 2-20 microns long and 0.3-0.9 microns wide. The pneumonia caused by Legionella infection is named Legionnaires' disease. So far, more than 70 species of Legionella have been discovered. All Legionella species found have been successfully isolated from the environment, of which about 30 are associated with the human respiratory tract. [0003] Of all species of Legionella, more than 90% of infections are caused by L. pneumophila. More than 90% of infections caused by Legionella pneumophila are caused by serotype 1 (L.pneumophilaserogroup 1). Legionella is mainly caused by the inhalation of aerosols containing Legionella by an infected person. When a person inhales an aerosol containing Legionella, the Legionella...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N5/071C12N15/85A01K67/027
CPCA01K67/0275A01K2207/12A01K2227/105A01K2267/0337C12N1/20C12N5/0686C12N15/85C12N2510/00
Inventor 张鹏唐春花邢宽何志健谢珍倪海钰周权何贵伦安雪茹李平江晓琴
Owner 南京实践医学检验有限公司
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