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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 and quantum dot technology, applied in the field of medical testing, can solve the problems of false positives, complicated operation steps, and high cost

Active Publication Date: 2017-02-15
湖北诺美华抗体药物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Example 1 Preparation of 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, and HN3-His fusion proteins

[0095] 1. Cloning of related genes

[0096] Bioinformatics analysis of I, II, and III human parainfluenza virus HN proteins (accession numbers in the NCBI protein database are AAC23946.1, BAA00739.1, and ACF28540.1), and the extracellular domains The peptide with the most abundant epitope, find its corresponding DNA coding sequence, and then optimize the codon according to the codon preference of E. coli, and introduce the restriction site NdeI at the 5'end and stop at the 3'end After the signal TAA and the restriction site XhoI are chemically synthesized, the full gene sequence is chemically synthesized (the full sequence synthesis is completed by GenScript Biotechnology Co., Ltd., and the artificially synthesized gene fragments are ...

Embodiment 2

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

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

[0113] Magnetic beads conjugated with anti-human parainfluenza virus HN protein polyclonal antibody IgG are used as solid-phase carrier, and quantum dot-labeled anti-human parainfluenza virus HN protein polyclonal antibody is used as the detection antibody. The detection is carried out 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 optimized selections were made for the particle size of the magnetic beads, as well as the EDC / NHS activator concentration, coupling antibody concentration, coupling time, and type of blocking agent.

[0114] 1.1 Selection of magnetic bead size

[0115] Select the carboxyl magnetic beads with particle...

Embodiment 3

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

[0129] 1. Optimization of reaction conditions for IgG reaction of mouse anti-type I human parainfluenza virus HN protein polyclonal antibody labeled with nano-carboxyl quantum dots:

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

[0131] The pH of the phosphate buffer in the labeling reaction was set to 5, 6, 7, 8, 9 respectively. The fluorescence intensity of the labeled product was measured by a full-spectrometer, and the effect of different pH values ​​on the coupling reaction was observed. Quantum dot-labeled polyclonal antibodies were determined The optimal pH of the reaction is 7.0-8.0. This experiment chooses pH7.4.

[0132] 1.2 Determination of the optimal labeling amount of carboxyl quantum dot-labeled antibody probe

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

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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 and a detection kit for detecting human parainfluenza virus antigen based on magnetic separation and quantum dot labeling, as well as a preparation and use method of the detection kit. Background technique [0002] Human parainfluenza virus (HPIV) is an important pathogen of acute respiratory infections in children. It is mainly transmitted by droplets and can also be transmitted through mucosal contact of the eyes, mouth or nose. The incidence is highest in infants and young children. Human parainfluenza virus infection has a global distribution and is a common pathogen of community-acquired respiratory infections. The incidence of human parainfluenza virus infection in children and children with acute respiratory infections is as high as 30-40%, which causes severe lower respiratory tract infections in children. , A pathogen second only to respirat...

Claims

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Application Information

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Patent Type & Authority Patents(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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