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Rapid detection method and kit for human parainfluenza virus based on magnetic separation and quantum dot labeling

A human parainfluenza virus, magnetic separation technology, applied in the field of medical detection, can solve the problems of complicated operation steps, high cost, and inability to be used as a clinical diagnosis method

Active Publication Date: 2016-01-27
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

Neither immunofluorescence method nor immunoenzyme method can perform one-step detection, and both have the disadvantages of complicated operation steps, professional operation, long detection time (more than 2 hours), and high cost.
The PCR method is fast, sensitive, and specific, and it is an important method for studying human parainfluenza virus infection. However, due to the high requirements for experimental equipment and operation of PCR, and the possibility of false positives, it cannot be used as a commonly used clinical diagnosis method in my country.

Method used

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  • Rapid detection method and kit for human parainfluenza virus based on magnetic separation and quantum dot labeling
  • Rapid detection method and kit for human parainfluenza virus based on magnetic separation and quantum dot labeling
  • Rapid detection method and kit for human parainfluenza virus based on magnetic separation and quantum dot labeling

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] The preparation of embodiment 1 rabbit and mouse anti-I, II and III type human parainfluenza virus HN protein polyclonal antibody IgG

[0094] (1) Preparation, purification and renaturation of recombinant HN1-His, HN2-His, HN3-His fusion proteins

[0095] 1. Cloning of related genes

[0096]Bioinformatics analysis was performed on the HN proteins of human parainfluenza virus types I, II, and III (the accession numbers in the NCBI protein database are AAC23946.1, BAA00739.1, and ACF28540.1, respectively), and the antigens in their extracellular domains were respectively obtained Find the corresponding DNA coding sequence for the peptide with the most abundant epitope, and then optimize its codon according to the codon preference of Escherichia coli, and introduce a restriction site NdeI at the 5' end and a termination signal at the 3' end After TAA and enzyme cutting site XhoI, chemically synthesize the whole gene sequence respectively (the whole sequence synthesis is h...

Embodiment 2

[0111] Example 2 Preparation of anti-human parainfluenza virus immune nano-magnetic beads

[0112] 1. Optimization of reaction conditions for anti-human parainfluenza virus HN protein polyclonal antibody coupled to magnetic beads:

[0113] Using magnetic beads coupled with anti-type I human parainfluenza virus HN protein polyclonal antibody IgG as a solid phase carrier, and quantum dot-labeled anti-human parainfluenza virus HN protein polyclonal antibody as a detection antibody, it is detected by the principle of double antibody sandwich method Type I human parainfluenza virus antigen, observe the coupling of magnetic 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.

[0114] 1.1 Selection of magnetic bead size

[0115] Select carboxyl magnetic beads with pa...

Embodiment 3

[0128] Example 3 Preparation of quantum dot-labeled anti-human parainfluenza virus nanoprobes

[0129] 1. Optimization of the IgG reaction conditions for nanocarboxyl quantum dot-labeled mouse anti-type I human parainfluenza virus HN protein polyclonal antibody IgG:

[0130] 1.1. Determination of the optimal labeling pH of the carboxyl quantum dot-labeled antibody probe

[0131] 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.

[0132] 1.2. Determination of the optimal labeling amount of carboxy quantum dot-labeled antibody probes

[0133] Set the ratio of quantum dot molar concentration to polyclonal antibody concen...

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Abstract

The invention discloses a rapid detection method and a kit for human parainfluenza virus based on magnetic separation and quantum dot labeling. The kit is composed of anti-human-parainfluenza-virus immunonanomagnetic beads with a I, II and III human parainfluenza virus enrichment function, anti-human-parainfluenza-virus nanoprobes labeled with quantum dots, quality control products and a PBST buffer solution. The quality control products comprise positive quality control products and negative quality control products. The positive quality control products are prepared after inactivated human parainfluenza virus is dried and combined on swabs. The negative quality control products are throat swabs of people who are determined to be negative in human parainfluenza virus. The detection method and the kit which can detect human parainfluenza virus antigens simply, rapidly and with high sensitivity are provided, and preparation and usage methods for the kit are provided.

Description

technical field [0001] The invention relates to the technical field of medical detection, in particular to a rapid detection method for detecting human parainfluenza virus 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] Human parainfluenza virus (HPIV) is an important pathogen of acute respiratory infection in children. It is mainly transmitted through droplets, and can also be transmitted through contact with mucous membranes of eyes, mouth or nose. The incidence rate is highest in infants and young children. Human parainfluenza virus infection has a global distribution and is a common pathogen of community-acquired respiratory tract infection. The incidence of human parainfluenza virus infection in children with acute respiratory tract infection is as high as 30-40%, which is the leading cause of severe lower respiratory tract infection in children. , secon...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/531G01N33/533
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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