Rapid detection method and kit for human mycoplasma pneumoniae based on magnetic separation and quantum dot labeling

A technology for mycoplasma pneumoniae and magnetic separation, which is applied in the field of medical detection and can solve the problems of low content of human mycoplasma pneumoniae, no clinical application, and low positive rate.

Active Publication Date: 2017-01-18
湖北诺美华抗体药物技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the publicly reported methods for detecting human Mycoplasma pneumoniae antigens are mainly double-antibody sandwich ELISA method, quantum dot labeling immunochromatography method, indirect immunofluorescence method, etc., but these methods have high sensitivity, but due to the clinical The content in specimens such as throat swabs is extremely low, resulting in a low positive rate of clinical detection, and has not been used in clinical practice at present

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  • Rapid detection method and kit for human mycoplasma pneumoniae based on magnetic separation and quantum dot labeling
  • Rapid detection method and kit for human mycoplasma pneumoniae based on magnetic separation and quantum dot labeling

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Experimental program
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Embodiment 1

[0078] Example 1 Preparation of Rabbit and Mouse Anti-human Mycoplasma Pneumoniae P1 Protein Polyclonal Antibody IgG

[0079] (1) Preparation and purification of recombinant P1-His fusion protein

[0080] 1. Cloning of related genes

[0081] Bioinformatics analysis was carried out on human Mycoplasma pneumoniae membrane protein P1 (the accession number in the NCBI protein database is AAK92040), to obtain the most abundant epitope peptide in its extracellular conserved domain, and to find its corresponding DNA coding sequence, According to the codon preference of Escherichia coli, the codon was optimized, and at the same time, a restriction site NdeI was introduced at the 5' end, a termination signal TAA and a restriction site XhoI were introduced at the 3' end, and the whole gene sequence (full sequence) was chemically synthesized. The synthesis was completed by GenScript Biotechnology Co., Ltd., and the artificially synthesized gene fragment was connected to the vector pUC57...

Embodiment 2

[0096] Example 2 Preparation of anti-mycoplasma pneumoniae immune nano-magnetic beads

[0097] 1. Optimization of reaction conditions for anti-mycoplasma pneumoniae polyclonal antibody-coupled magnetic beads:

[0098] Using magnetic beads coupled with anti-mycoplasma pneumoniae polyclonal antibody as a solid-phase carrier, and quantum dot-labeled polyclonal antibody against mycoplasma pneumoniae P1 protein as a detection antibody, the human mycoplasma pneumoniae antigen was detected by the principle of double-antibody sandwich method, and the magnetic field was observed. Coupling of beads and polyclonal antibodies. A series of optimization options were carried out on the particle size of the magnetic beads, as well as the concentration of EDC / NHS activator, the concentration of conjugated antibody, the coupling time, and the type of blocking agent.

[0099] 1.1 Selection of magnetic bead size

[0100] Select carboxyl magnetic beads with a particle size of 50nm, 180nm, 350nm,...

Embodiment 3

[0111] Example 3 Preparation of quantum dot-labeled anti-mycoplasma pneumoniae nanoprobes

[0112] 1. Optimization of the IgG reaction conditions for the nano carboxyl quantum dot-labeled mouse anti-human Mycoplasma pneumoniae P1 protein polyclonal antibody IgG:

[0113] 1.1. Determination of the optimal labeling pH of the carboxyl quantum dot-labeled antibody probe

[0114] The pH of the phosphate buffer in the labeling reaction was set to 5, 6, 7, 8, and 9 respectively, and the fluorescence intensity of the labeled product was measured with a full spectrometer, and the influence of different pH values ​​on the coupling reaction was observed, and the quantum dot labeled polyclonal antibody was determined. The optimum pH for the reaction is 7.0-8.0. This experiment chooses pH7.4.

[0115] 1.2. Determination of the optimal labeling amount of carboxy quantum dot-labeled antibody probes

[0116] Set the ratio of quantum dot molar concentration to polyclonal antibody concentrat...

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Abstract

The invention provides a method for detection of human chlamydia pneumoniae antigen based on magnetic resolution and quantum dot labelling. The method comprises the following steps: (1) preparing anti-human chlamydia pneumoniae immune nano magnetic beads; (2) preparing quantum dot labelled anti-human chlamydia pneumoniae nanoprobes; (3) dissolving a sample to be detected with a PBST buffer solution, adding the anti-human chlamydia pneumoniae immune nano magnetic beads into the dissolved solution, carrying out magnetic separation after sufficient mixing and reaction, washing with the PBST buffer solution, adding the quantum dot labelled anti-human chlamydia pneumoniae nanoprobes into the obtained precipitate, carrying out magnetic separation after a reaction, washing with the PBST buffer solution, and detecting the fluorescence value with a fluorescence micro-plate reader. The method is accurate, fast, and high in sensitivity, and has very high practical value in the aspects of clinical diagnosis, etiological differentiation, epidemiological surveys and the like of human chlamydia pneumoniae.

Description

technical field [0001] The invention relates to the technical field of medical detection, in particular to a rapid detection method for detecting human mycoplasma pneumoniae antigen based on magnetic separation and quantum dot labeling, a detection kit, and a method for preparing and using the detection kit. Background technique [0002] Mycoplasma pneumoniae (Mycoplasma pneumoniae, Mp) is the pathogen of human mycoplasma pneumonia, mainly transmitted through droplets, with an incubation period of 2-3 weeks, and the incidence rate is highest in adolescents. The incidence of Mp infection in children with pneumonia is as high as 10-30%. In recent years, it has gradually become one of the main pathogens of children with respiratory diseases. The disease can easily lead to respiratory infections such as pharyngitis and tonsillitis, and may even cause multiple organ injuries such as meningitis, hepatitis, and myocarditis at the same time, and may even lead to death in severe case...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68
Inventor 胡征杨波董俊
Owner 湖北诺美华抗体药物技术有限公司
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