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Method for preparing cell substrate, method for preparing kit and joint detection method

A kit and cell technology, applied in the field of immunoassay, can solve the problems of high specificity, long time-consuming separation and culture method, unfavorable promotion, etc., achieve the effect of shortening the time and avoiding the interference of non-specific fluorescence

Pending Publication Date: 2021-12-17
北京英诺特生物技术股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among them, the isolation and culture method has the disadvantages of long time-consuming, low sensitivity and high technical requirements; in the determination of antigens, although the evidence of infection can be clearly identified and rapid detection can be achieved, its sensitivity is too low; while in the detection of specific antibodies High specificity and low sensitivity lead to inability to assist clinical diagnosis in time, thus making patients unable to receive effective treatment in time; although molecular biology methods have the advantages of high sensitivity and rapid detection, they are not conducive to the high requirements on the experimental environment. to promote

Method used

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  • Method for preparing cell substrate, method for preparing kit and joint detection method
  • Method for preparing cell substrate, method for preparing kit and joint detection method
  • Method for preparing cell substrate, method for preparing kit and joint detection method

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preparation example Construction

[0094] The preparation of a cell suspension infected with non-bacteria involves the following steps:

[0095] In step S21, at least two kinds of passaged cells (for example, the passaged cells obtained in step S12) among various pathogens are digested and separated with trypsin at a concentration ranging from 0.1% to 0.5%, preferably at a concentration of 0.25%. The at least two kinds of passaged cells are detached from the bottom of the culture vessel (for example, the bottom of the culture bottle), and stop digestion and separation when they appear in a three-dimensional shape, and suck out the trypsin;

[0096] In step S22, the digested and separated at least two types of passaged cells are respectively placed in 5% CO with a humidity ≥ 90%, preferably a humidity of 96% and a temperature of 37°C. 2 Adsorption in the incubator for 1h to 3h, preferably 2h;

[0097] In step S23, supplement the adsorbed at least two kinds of passaged cells with fetal bovine serum medium in a c...

Embodiment 1

[0119] 1. The method for preparing the cell substrate for joint detection of respiratory infection comprises the following steps:

[0120] (1) Subculture of cells:

[0121] Method 1 Pathogen-infected cell culture: Add 10% fetal bovine serum MDEM medium to Hep-2, MDCK, MK2, and A549 cells to be fixed, and then place them at a temperature of 37°C, a humidity of 40%, and CO 2 Concentration of 5% CO 2 Cultivate in the incubator for 48 hours to recover; then digest and separate with 0.25% trypsin, aspirate the trypsin and count 6 million / T75, and place it again in 5% CO with a temperature of 37°C and a humidity of 90%. 2 Cultured in an incubator for 24 hours for subculture.

[0122] Method 2: Cultivate Legionella pneumophila LP cells: After the LP special medium powder is dissolved, carry out autoclaving at 121°C for 15 minutes, pour it into a plate after cooling, and make about 15ml of medium in each plate. After the medium solidifies, Inoculate 50uL of LP bacteria and place in...

Embodiment 2

[0143] Common pathogens of enteric infection include rotavirus RV, adenovirus ADV, astrovirus Astrovirus, and enterovirus 71 EV71.

[0144] 1. The preparation method of the cell substrate for joint detection of intestinal infection comprises the following steps:

[0145] (1) Subculture of cells:

[0146] Method 1 Pathogen-infected cell culture: Add 10% fetal bovine serum MDEM medium to the MA-104, ADV, MK2, and Caco-2 cells to be fixed, and then place them in a 5% medium with a temperature of 37°C and a humidity of 98%. CO 2 Cultivate in the incubator for 48 hours to recover; then digest and separate with 0.25% trypsin, aspirate the trypsin and count 6 million / T75, and place it again in 5% CO with a temperature of 37°C and a humidity of 98%. 2 Cultured in an incubator for 24 hours for subculture.

[0147] (2) Pathogen-infected cells:

[0148] To inoculate and infect the cells by selecting half of the virus seed amount of TCID50, and digest and separate the cells before the...

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Abstract

The invention discloses a method for preparing a cell substrate for multi-pathogen joint detection, a method for preparing a kit for multi-pathogen joint detection and a multi-pathogen joint detection method, and belongs to the field of immunodetection. The method for preparing a cell substrate for multi-pathogen joint inspection comprises the following steps of: respectively carrying out cell subculture on multiple pathogens; respectively preparing cell suspensions from at least two kinds of passage cells; and respectively applying the cell suspensions into different glass slide holes on the same glass slide, and fixing the cells in the different glass slide holes under the same condition after the cells are adsorbed. The multiple pathogens comprise at least two pathogenic microorganisms in any one pathogen selected from a respiratory system infection pathogen, a blood system infection pathogen, a digestive system infection pathogen and a reproductive system infection pathogen. The same condition is that absolute ethyl alcohol is used for fixing for the same time under the same temperature condition, and drying is performed for the same time in the same humidity environment with the humidity less than 50% to form the cell substrate.

Description

technical field [0001] The invention relates to the technical field of immune detection, in particular to a method for preparing a cell substrate for joint inspection of multiple pathogens, a method for preparing a kit for joint inspection of multiple pathogens, and a method for joint inspection of multiple pathogens. Background technique [0002] Most infections are usually caused by pathogens such as viruses, bacteria, mycoplasma, and chlamydia. During treatment, it is necessary to clarify the type of pathogen causing the infection, which is the prerequisite for effective treatment. Since the same type of pathogen can cause multiple clinical symptoms, the same clinical manifestation may be caused by multiple pathogens. For example, in patients with respiratory system infection, the proportion of co-infection accounts for about 20%, while a single virus or The detection of pathogens such as bacteria can no longer meet the needs of clinical diagnosis. [0003] Therefore, jo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28
CPCG01N1/2813
Inventor 张秀杰吴吟妮张晓刚刘洋刘雪敏
Owner 北京英诺特生物技术股份有限公司
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