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

A group A streptococcus and quantum dot technology, applied in the field of medical detection, can solve the problems of detection sensitivity, low sensitivity of colloidal gold method, and inability to be used as a clinical diagnosis method

Active Publication Date: 2017-02-01
湖北诺美华抗体药物技术有限公司
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  • Abstract
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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 mainly used to detect streptococcus-specific rRNA sequences. Its detection is rapid, sensitive and specific, and it is an important method for studying group A streptococcal infection. It cannot be used as a commonly used clinical diagnosis method in my country
At present, the method for detecting human group A streptococcal antigen is mainly colloidal gold method to detect its C polysaccharide antigen, but the sensitivity of colloidal gold method is low, and the quality requirements of sample materials are high. At the same time, the sample must be lysed before detection. To release C polysaccharide, and the amount of cell lysate added has a greater impact on the detection sensitivity

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1 Preparation of rabbit and mouse anti-human group A streptococcus 1, 3, 5, 6, 24 type M protein polyclonal antibody IgG

[0085] (1) Preparation and purification of recombinant M-His fusion protein

[0086] 1. Cloning of related genes

[0087] Bioinformatics analysis of human group A streptococcus type 1, 3, 5, 6, 24 M protein (accession numbers in the NCBI protein database are AAK34694, NP_802987, WP_023079553, EZN36963, and WP_027968818), and obtain their N-terminals respectively The type-specific sequence (hypervariable region) contains extracellular antigen epitopes and does not cross-react with other antigens and antibodies; find the corresponding gene coding sequence of the five peptides, press M1-M3-M5 -The sequence of M6-M24 is spliced ​​and the sequence is codon-optimized according to the preference of codons in E. coli, and restriction digestion is introduced at the 5'end and 3'end of the codon-optimized recombinant gene sequence Site and chemically synthe...

Embodiment 2

[0101] Example 2 Preparation of anti-human group A streptococcus immune nanomagnetic beads

[0102] 1. Optimization of reaction conditions for coupling of anti-human group A streptococcus polyclonal antibody to magnetic beads:

[0103] Magnetic beads conjugated with anti-human group A streptococcus 1, 3, 5, 6, 24 type M protein polyclonal antibody as solid phase carrier, quantum dot labeled anti-human group A streptococcus 1, 3, 5, 6 , 24 type M protein polyclonal antibody is used as the detection antibody to detect human group A streptococcus antigens through the principle of double antibody sandwich method, and 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.

[0104] 1.1 Selection of magnetic bead size

[0105] Select the carboxyl magnetic beads with parti...

Embodiment 3

[0116] Example 3 Preparation of quantum dot-labeled anti-human group A streptococcus nanoprobe

[0117] 1. Optimization of reaction conditions for IgG reaction of mouse anti-human group A streptococcus type 1, 3, 5, 6, 24 M protein polyclonal antibody labeled with nano-carboxyl quantum dots:

[0118] 1.1. Determination of the optimal labeling pH for carboxyl quantum dot labeled antibody probe

[0119] 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.

[0120] 1.2 Determination of the optimal labeling amount of carboxyl quantum dot-labeled antibody probe

[0121] Set the ratio of the molar concentration of quantum dots to the concentration of polycl...

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Abstract

The invention provides a method for detecting a human group A streptococci antigen based on magnetic separation and quantum dot labeling. The method includes the steps: (1) preparing anti-human group A streptococci immune nano magnetic beads; (2) preparing quantum dot labeled anti-human group A streptococci nano probe; and (3) after dissolving a to-be-tested sample in a PBST buffer solution, adding the anti-human group A streptococci immune nano magnetic beads to the dissolved solution, fully mixing, carrying out a reaction, then carrying out magnetic separation, washing with a PBST buffer solution, adding the quantum dot labeled anti-human group A streptococci nano probe to the obtained precipitate, carrying out a reaction, then carrying out magnetic separation, washing with a PBST buffer solution, and then detecting a fluorescent value by using a fluorescence microplate reader. The accurate, rapid and high-sensitivity method for detecting human group A streptococci has quite high practical value in clinical diagnosis, etiology identification and epidemiological investigation of human M1, M3, M5, M6 and M24 serotype group A streptococci.

Description

Technical field [0001] The invention relates to the technical field of medical detection, in particular to a rapid detection of human group A streptococci (GAS) (M1, 3, 5, 6, 24 serotype) antigens based on magnetic separation and quantum dot labeling Method and detection kit, as well as preparation and use method of the detection kit. Background technique [0002] Streptococcus is widely distributed in nature and animals, and can live in human nasopharynx and intestines for a long time. It is a gram-positive bacteria, spherical or oval, aerobic or facultative anaerobe, and has high nutritional requirements. There are many kinds of streptococci. According to their hemolytic ability, they are divided into alpha, beta, and gamma hemolytic streptococci, also known as type A, type B, and type C hemolytic streptococci. According to the different polysaccharides (C antigen) contained in the cell wall of streptococcus, it is divided into 20 groups such as A to V (including I and J miss...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569
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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