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

A Streptococcus pneumoniae, magnetic separation technology, applied in the field of medical testing, can solve the problems of high quality requirements, false positives, and cannot 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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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Both immunofluorescence and immunoenzyme methods have disadvantages such as complex operation steps, professional operation, long detection time (more than 2 hours), and high cost
The PCR method is fast, sensitive, and specific, and is an important method for studying Streptococcus pneumoniae 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.
The target antigen of colloidal gold immunochromatography is C polysaccharide antigen, but the sensitivity of colloidal gold method is low, the quality requirements of sample materials are high, and there are also defects such as cross-reaction with other streptococci such as streptococcus moderatee

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Example 1 Preparation of rabbit and mouse anti-human Streptococcus pneumoniae Fam1PspA, Fam2PspA protein polyclonal antibody IgG

[0091] (1) Preparation and purification of recombinant PspA1-His and PspA2-His fusion proteins

[0092] 1. Cloning of related genes

[0093]Bioinformatic analysis was performed on the Fam1PspA and Fam2PspA proteins of human Streptococcus pneumoniae (the accession numbers in the NCBI protein database are AAF27703 and AAF27712), respectively, to obtain the peptides with the most abundant antigenic epitopes in their extracellular domains, and to find their corresponding At the same time, 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 (the whole sequence synthesis was completed by GenScript Biotechnology Co., Ltd. , the artificially synthesized gene fragments were connected to the vector pUC57 at the time of deli...

Embodiment 2

[0106] Example 2 Preparation of anti-human Streptococcus pneumoniae immune nano-magnetic beads

[0107] 1. Optimization of reaction conditions for anti-human Streptococcus pneumoniae Fam1PspA protein polyclonal antibody coupled to magnetic beads:

[0108] Using magnetic beads coupled with rabbit anti-human Streptococcus pneumoniae Fam1PspA protein polyclonal antibody IgG as a solid phase carrier, and quantum dot-labeled mouse anti-human Streptococcus pneumoniae PspA protein polyclonal antibody as a detection antibody, it is detected by the principle of double-antibody sandwich method Human Streptococcus pneumoniae subtype strain Sp6B 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.

[0109] 1.1 Selection of magnetic ...

Embodiment 3

[0123] Example 3 Preparation of quantum dot-labeled anti-human Streptococcus pneumoniae nanoprobe

[0124] 1. Optimization of IgG reaction conditions for nano carboxy quantum dot-labeled mouse anti-human Streptococcus pneumoniae Fam1PspA protein polyclonal antibody IgG:

[0125] 1.1. Determination of the optimal labeling pH of the carboxyl quantum dot-labeled antibody probe

[0126] 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.

[0127] 1.2. Determination of the optimal labeling amount of carboxy quantum dot-labeled antibody probes

[0128] Set the ratio of quantum dot molar concentration to polyclonal antibody co...

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Abstract

The invention discloses a rapid detection method and a kit for streptococcus pneumoniae based on magnetic separation and quantum dot labeling. The kit is composed of anti-streptococcus-pneumoniae immunonanomagnetic beads with a streptococcus pneumoniae enrichment function, anti-streptococcus-pneumoniae 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 streptococcus pneumoniae is dried and combined on swabs. The negative quality control products are throat swabs of people who are determined to be negative in streptococcus pneumoniae. The detection method and the kit which can detect streptococcus pneumoniae 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 Streptococcus 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] Human Streptococcus pneumoniae (Streptococcuspneumoniae, Sp) is an important pathogen of respiratory tract infection in children. It enters the respiratory tract mainly through droplets, parasitizes the nasopharynx of the human body, or invades parts of the human body that are not easy to remove, causing a series of diseases, such as lobar pneumonia, meningitis, bronchitis, otitis media, etc. It is the leading pathogen with high morbidity and mortality in all age groups worldwide. Among them, in developing countries, infants, the elderly, and immunocompromised populations are particularly serious. Streptococcus pneumoniae was first isol...

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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