Human group A streptococci quantum dot immunochromatography detection card, preparation method and applications

A group A streptococcus, immunochromatographic technology, applied in the field of medical detection, can solve the problems of detection sensitivity, prone to 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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  • 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 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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  • Human group A streptococci quantum dot immunochromatography detection card, preparation method and applications
  • Human group A streptococci quantum dot immunochromatography detection card, preparation method and applications
  • Human group A streptococci quantum dot immunochromatography detection card, preparation method and applications

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Experimental program
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Effect test

preparation example Construction

[0081] 1. Preparation of conjugated pads

[0082] (1) Preparation and purification of recombinant M-His fusion protein:

[0083] Bioinformatic analysis was performed on human group A streptococcal type 1, 3, 5, 6, and 24 M proteins, and their N-terminal type-specific sequences (hypervariable regions) contained extracellular antigenic epitopes and were not associated with other antigens. The peptides that cross-react with the antibody are marked as M1, M3, M5, M6 and M24 respectively; find the gene coding sequence corresponding to the above five peptides, and sequence them in the order of M1-M3-M5-M6-M24 Splicing and codon-optimizing the sequence according to the codon preference in Escherichia coli, introducing enzyme cutting sites at the 5' and 3' ends of the above-mentioned codon-optimized recombinant gene sequence and chemically synthesizing the entire gene sequence , and marked as m at the same time; its gene sequence is shown in the sequence table; the gene sequence is c...

Embodiment 1

[0122] Embodiment 1 (preparation embodiment)

[0123] Conjugate pad preparation

[0124] (1) Preparation and purification of recombinant M-His fusion protein

[0125] 1. Cloning of related genes

[0126] Bioinformatic analysis was performed on human group A Streptococcus type 1, 3, 5, 6, and 24 M proteins (the accession numbers in the NCBI protein database are AAK34694, NP_802987, WP_023079553, EZN36963, and WP_027968818) to obtain their N-terminal types Peptides that contain extracellular antigenic epitopes in the specific sequence (hypervariable region) and do not cross-react with other antigens and antibodies are marked as M1, M3, M5, M6 and M24 respectively; find the five peptides one by one The corresponding gene coding sequence is spliced ​​in the order of M1-M3-M5-M6-M24 and the sequence is codon-optimized according to the codon preference in Escherichia coli. The 5' end and 3' end of the sequence are introduced with enzyme cutting sites and the whole gene sequence i...

Embodiment 2

[0152] Embodiment 2 (preparation embodiment)

[0153] Preparation of sample pads

[0154] Prepare sample pad treatment solutions with different formulations, observe the release effect of quantum dot-labeled antibodies, and optimize through multiple orthogonal experiments to obtain the optimal sample pad treatment solution formulation (that is, described in the present invention). Take a piece of glass cellulose membrane, soak it in the sample pad treatment solution for at least 3 hours, then place it in a biological safety cabinet at 37°C, ventilate and dry it, and cut it into a size of 4cm*2.5cm / strip to prepare the sample pad , Store in airtight and dry place at 25°C. Tests have proved that the use of the sample pad greatly improves the release rate of the quantum dot-labeled antibody on the binding pad and achieves a better application effect.

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Abstract

The invention discloses a human group A streptococci quantum dot immunochromatography detection card, a preparation method and applications. The detection card comprises a base plate, a sample pad, a combination pad, a detection layer and a water absorption pad. The combination pad is coated with anti-human group A streptococci nano probes labeled with quantum dots. The detection layer is composed of a solid phase nitrocellulose membrane with a detection line and a quality control line. The detection line is coated with mouse-anti-human group A streptococci M protein polyclonal antibodies. The quality control line is coated with anti-rabit IgG. The detection layer is pasted on the base plate. The combination pad and the water absorption pad are pasted with the detection layer and the base plate respectively. The sample pad is arranged on the combination pad, overlaps with part of the combination pad and is pasted with the combination pad and the base plate. The human group A streptococci (M1, 3, 5, 6, 24 serotype) quantum dot immunochromatography detection card with advantages of simple operation, rapid detection, quantification, high sensitivity and the like, a preparation method and applications are provided.

Description

technical field [0001] The invention relates to the technical field of medical detection, in particular to a human group A streptococcus (M1, 3, 5, 6, 24 serotype) quantum dot immunochromatographic detection card and a preparation method and application thereof. Background technique [0002] Streptococcus is widely distributed in nature and animals, and can live in the nasopharynx and intestinal tract of humans for a long time. Gram-positive bacteria, spherical or oval, aerobic or facultative anaerobic bacteria, with high nutritional requirements. There are many kinds of streptococci, which are divided into α, β, and γ hemolytic streptococci according to their hemolytic ability, also known as type A, type B, and type C hemolytic streptococci. According to the different polysaccharides (C antigens) contained in the streptococcal cell wall, they are divided into 20 families such as A~V (including I and J missing), (Lancefield typing). 90% of streptococci that are pathogenic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/532
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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