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Quantitative detection method and kit for rapid diagnosis of human respiratory pathogens

A rapid diagnosis and respiratory technology, applied in the field of pathogen diagnosis, can solve the problems that the staining efficiency is greatly affected by the permeability rate, the cell culture method has a long cycle, and the transportation and storage are easy to be infected, so as to achieve high sensitivity, fast detection and time saving. Effect

Pending Publication Date: 2020-06-05
天津美瑞特医疗科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the gold standard for the diagnosis of respiratory virus infection is still the isolation and culture of the virus, but the cell culture method has a long cycle, transportation and storage are prone to infection, and the practical application is limited.
The direct immunofluorescence method on slides needs to judge the result by visually observing the number of green fluorescent cells, so the judgment of the result is subjective, and only a single fluorescein-labeled antibody can be used at a time, so only one pathogen can be identified at a time. Low detection efficiency
[0005] The technologies and products currently on the market that use flow cytometry to detect respiratory pathogens mainly use solid liquid to fix the cells, and use a permeabilizer to perform fluorescent staining on the virus in the cells. The dyeing efficiency is greatly affected by the permeability and cannot be quantified. analyze

Method used

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  • Quantitative detection method and kit for rapid diagnosis of human respiratory pathogens
  • Quantitative detection method and kit for rapid diagnosis of human respiratory pathogens
  • Quantitative detection method and kit for rapid diagnosis of human respiratory pathogens

Examples

Experimental program
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Effect test

Embodiment 1

[0050] Example 1: Rapid Diagnosis and Detection Kit for Human Respiratory Pathogens

[0051] Including human respiratory pathogen RSV antibody capture microspheres, PE-labeled detection antibody reagents, RSV recombinant protein standards, flow cytometer calibration microspheres, sample buffer, microsphere buffer, lysis buffer, washing solution. The respiratory pathogens described therein also include respiratory syncytial virus (RSV), influenza A virus (IA), influenza B virus (IB), adenovirus (Adv), parainfluenza virus type 1, parainfluenza virus type 2 and At least one of parainfluenza virus type 3, metapneumovirus, boca virus, coronavirus, rhinovirus, enterovirus, mycoplasma or chlamydia.

Embodiment 2

[0052] Example 2: Preparation of Human Respiratory Pathogen Detection Kit

[0053] The present embodiment is the preparation of a single RSV antibody detection kit for human respiratory pathogens, which is carried out according to the following steps:

[0054] 1. Preparation of RSV antibody capture microspheres

[0055] 1) Solution preparation

[0056] Preparation of buffer A: Weigh 0.96g of disodium hydrogen phosphate, add 600mL of deionized water to dissolve, adjust the pH to 6.0, add water to make up to 800mL, and filter with a 0.45um water-based filter membrane;

[0057] Preparation of buffer B: Weigh 7.5g of glycine, add 800mL of water to dissolve, adjust the pH to 7.5, add water to make up to 1000mL, and filter with 0.45um water-based filtration membrane;

[0058] 20mg / ml 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC) solution was prepared; 1g of EDC was weighed, dissolved in 50mL of water, and filtered with a 0.45um water-based filtration membrane;...

Embodiment 3

[0075] Example 3: Rapid Diagnosis and Detection Method of Human Respiratory Pathogens

[0076] This example is a method for detecting a single pathogen (RSV) using the kit prepared in Example 2, which is carried out according to the following steps:

[0077] 1. Drawing of the standard curve: Take the human recombinant RSV antibody standard, add 2 mL of sample buffer, let it stand at room temperature for 15 minutes, and use a pipette to gently mix the standard. At this time, the concentration of the standard is 5000 viruses per ml. Quantity; take 10 flow sampling tubes and mark the serial dilution multiples as 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64, 1 / 128 / , 1 / 256 , 0; add 200μL of sample buffer to each tube, take 200ul of liquid from the standard tube to 1 / 2 tube, pipette and mix well and take 200μL of liquid to 1 / 4 tube, and so on to 1 / 256 tube; Take 50 ul of the RSV antibody capture microspheres prepared in Example 2, centrifuge at 200 g for 5 min after mixing, aspirate the superna...

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Abstract

The invention discloses a quantitative detection method and a kit for rapid diagnosis of human respiratory pathogens. The kit is composed of: at least one human respiratory tract pathogen antibody capture microsphere, a PE-labeled detection antibody, a pathogen protein standard substance, a flow cytometry correction microsphere, a microsphere buffer solution, a cell lysis solution, a sample diluent and a cleaning solution; the kit is simple, convenient, rapid and automatic to operate, high in detection result sensitivity, wide in detection range and good in repeatability, quantitative detection of various pathogens in human respiratory tract epithelial cells and body fluid can be achieved, detection objects can be flexibly combined, and the infection condition of the pathogens can be reflected more truly.

Description

technical field [0001] The invention belongs to the technical field of pathogen diagnosis, and in particular relates to a quantitative detection method and kit for rapid diagnosis of human respiratory pathogens, as well as preparation and use methods of the kit. Background technique [0002] Respiratory tract infection is the most common infectious disease in my country, most of which are caused by respiratory viruses, which can be divided into upper respiratory tract infection and lower respiratory tract infection. Influenza virus (IFV), parainfluenza virus (PIV), adenovirus (HAdV), metapneumovirus (MPV), coronavirus (CoV), respiratory syncytial virus (RSV), rhinovirus (RhV), enterovirus ( EV), Boca virus (HBoV), Mycoplasma pneumoniae, and Chlamydia pneumoniae are common respiratory pathogens. Therefore, rapid detection of pathogen types plays an important role in timely diagnosis of diseases, rational use of antibiotics and antiviral drugs, and prevention of infection. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/58G01N33/569G01N33/543G01N33/531G01N33/533
CPCG01N33/582G01N33/56983G01N33/56927G01N33/56933G01N33/54313G01N33/531G01N33/533G01N2333/135G01N2333/11G01N2333/115G01N2333/075G01N2333/085G01N2333/165G01N2333/015G01N2333/095G01N2469/10
Inventor 洪建
Owner 天津美瑞特医疗科技有限公司
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