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Rapid human respiratory syncytial virus detection method and kit based on magnetic separating and quantum dot labeling

A syncytial virus and magnetic separation technology, applied in the field of medical detection, can solve the problems of numerous operation steps, long detection time, and high detection cost

Active Publication Date: 2016-02-10
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

At present, the publicly reported methods for detecting human respiratory syncytial virus antigens are mainly double-antibody sandwich ELISA method, colloidal gold immunochromatography method, indirect immunofluorescence method, etc., but these methods may have many operation steps, long detection time, or There are problems such as low detection sensitivity and high detection cost

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  • Rapid human respiratory syncytial virus detection method and kit based on magnetic separating and quantum dot labeling
  • Rapid human respiratory syncytial virus detection method and kit based on magnetic separating and quantum dot labeling
  • Rapid human respiratory syncytial virus detection method and kit based on magnetic separating and quantum dot labeling

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1 Preparation of Rabbit and Mouse Anti-Human Respiratory Syncytial Virus NP Protein Polyclonal Antibody IgG

[0081] (1) Preparation and purification of recombinant NP-His fusion protein

[0082] 1. Cloning of related genes

[0083] The human respiratory syncytial virus nucleoprotein NP (the accessionnumber in the NCBI protein database is AAB59852) was analyzed by bioinformatics to obtain the peptide with the most abundant antigenic epitope in its conserved domain, find its corresponding DNA coding sequence, and then According to the codon preference of Escherichia coli, the codon was optimized, and the whole gene sequence was chemically synthesized after introducing the restriction site NdeI at the 5' end, the termination signal TAA and the restriction site XhoI at the 3' end (full sequence synthesis It was completed by GenScript Biotechnology Co., Ltd., and the artificially synthesized gene fragment was connected to the vector pUC57 at the time of delivery), ...

Embodiment 2

[0098] Example 2 Preparation of anti-human respiratory syncytial virus immune nano-magnetic beads

[0099] 1. Optimization of reaction conditions for anti-human respiratory syncytial virus polyclonal antibody coupled to magnetic beads:

[0100] Using magnetic beads coupled with anti-human respiratory syncytial virus NP protein polyclonal antibody as a solid phase carrier, and quantum dot-labeled anti-human respiratory syncytial virus NP protein polyclonal antibody as a detection antibody, the detection of human Respiratory syncytial 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.

[0101] 1.1 Selection of magnetic bead size

[0102] Carboxylated magnetic beads with a particle size of 50nm, 180nm, 350nm, 1150n...

Embodiment 3

[0113] Example 3 Preparation of quantum dot-labeled anti-human respiratory syncytial virus nanoprobes

[0114] 1. Optimization of the reaction conditions for the IgG reaction of the mouse anti-human respiratory syncytial virus NP protein polyclonal antibody labeled with nano carboxyl quantum dots:

[0115] 1.1. Determination of the optimal labeling pH of the carboxyl quantum dot-labeled antibody probe

[0116] 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.

[0117] 1.2. Determination of the optimal labeling amount of carboxy quantum dot-labeled antibody probes

[0118] Set the ratio of quantum dot molar concentrati...

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Abstract

The invention provides a human respiratory syncytial virus antigen detection method based on magnetic separating and quantum dot labeling. The method comprises the steps that 1, immune nano magnetic beads resisting a human respiratory syncytial virus are prepared; 2, quantum-dot-labeled nano-probes resisting the human respiratory syncytial virus are prepared; 3, after a sample to be detected is dissolved with a sample processing solution, the immune nano magnetic beads resisting the human respiratory syncytial virus are added in a dissolved solution, magnetic separating is performed after full mixing and reacting are performed, washing is performed with a PBST buffer solution, the quantum-dot-labeled nano-probes resisting the human respiratory syncytial virus are added in obtained precipitates, magnetic separating is performed after reacting is performed, and after washing is performed with the PBST buffer solution, a fluorescence value is detected with a fluorescence microplate reader. Accordingly, the accurate, rapid and high-sensitivity method for detecting the human respiratory syncytial virus is established, and the very high practical value in the aspects such as clinical diagnosis, etiology identification and epidemiological investigation of the human respiratory syncytial virus is achieved.

Description

technical field [0001] The invention relates to the technical field of medical detection, in particular to a rapid detection method for detecting human respiratory syncytial 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] Respiratory syncytial virus (RespiratorySyncytialVirus, RSV) is an RNA virus that belongs to Paramyxoviridae. The disease is transmitted by air droplets and close contact. More common in newborns and infants under 6 months. The incubation period is 3 to 7 days. Infants and young children have severe symptoms, such as high fever, rhinitis, pharyngitis and laryngitis, and later manifest as bronchiolitis and pneumonia. A small number of sick children may be complicated by otitis media, pleurisy and myocarditis. After infection in adults and older children, the main manifestation is upper respiratory tract infection. [0003] Accordin...

Claims

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Application Information

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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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