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Preparation method of latex microsphere immunochromatographic test paper based on Klebsiella pneumoniae surface protein

A Klebsiella pneumoniae and surface protein technology, applied in the direction of anti-bacterial immunoglobulin, immunoglobulin, chemical instruments and methods, etc., can solve problems such as difficult to meet rapid identification, affect the effect of amplification, and take a long time , to achieve the effects of low preparation cost, strong capture power and low cost

Active Publication Date: 2021-01-29
HUBEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003]At present, the method for detecting the pathogen in the respiratory tract is mainly based on the traditional method, that is, the separation and identification method. Difficult to meet the needs of rapid identification; the PCR technology developed in recent years is a fast, sensitive, and specific technology, but at present this technology still relies on the pre-enrichment step of the traditional method, and the enrichment solution often There are also PCR inhibitors, which affect the effect of amplification
At the same time, this technology also requires professional testing equipment, which is not suitable for bedside testing

Method used

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  • Preparation method of latex microsphere immunochromatographic test paper based on Klebsiella pneumoniae surface protein
  • Preparation method of latex microsphere immunochromatographic test paper based on Klebsiella pneumoniae surface protein
  • Preparation method of latex microsphere immunochromatographic test paper based on Klebsiella pneumoniae surface protein

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Embodiment 1

[0059]Preparation of Klebsiella pneumoniae surface protein (ABC+fepA) antibody:

[0060]1.1) Cloning of Klebsiella pneumoniae Abcfep fusion gene

[0061]Obtain Klebsiella pneumoniae surface proteins ABC transporter substrate-binding protein and FepA (the accession numbers in the NCBI protein database are WP_009653190 and WP_012068422 respectively) peptides with the most abundant epitopes in the extracellular domain and find the gene coding sequence Optimize its gene coding sequence, and connect these two sequences with the coding sequence of flexible connecting peptide (ggsggsggsggs) to form a fusion gene. At the same time, at the 5'end of the fusion gene, the restriction site NdeI was introduced, and the termination signal TAA and restriction site BamHI were introduced into the 3'end of the fusion gene to chemically synthesize the entire gene sequence, which was recorded as Abcfep. The full sequence of the gene and the encoded amino acid sequence are shown in the sequence table. Specific...

Embodiment 2

[0090]Preparation of Klebsiella pneumoniae surface protein (glpQ+mltD) antibody:

[0091]2.1) Cloning of Klebsiella pneumoniae Glpmlt fusion gene

[0092]Obtain Klebsiella pneumoniae surface proteins GlpQ and mltD (the accession numbers in the NCBI protein database are WP_004214637 and WP_004210407, respectively) peptides with the most abundant epitopes in the extracellular domain and find the gene coding sequence to optimize its gene coding The two sequences are connected with the coding sequence of rigid linking peptide (eaaakeaaak) to form a fusion gene. At the same time, at the 5'end of the fusion gene, the restriction site NdeI was introduced, and the termination signal TAA and restriction site BamHI were introduced into the 3'end of the fusion gene, and the complete gene sequence was chemically synthesized, which was recorded as Glpmlt. The full sequence of the gene and the encoded amino acid sequence are shown in the sequence table. Specifically, the protein sequences encoded by th...

Embodiment 3

[0122]Preparation of latex microsphere marker for Klebsiella pneumoniae surface protein (ABC+fepA) antibody:

[0123]3.1) Activation of latex microspheres

[0124]Take 1mL of 10% red carboxylated polystyrene latex microspheres (100nm) solution, add 9mL 2-(N-morpholinyl)ethanesulfonic acid (MES) buffer (0.1mol / L MES, pH8.5 ) And mix well; prepare 10mg / mL N-hydroxysuccinimide (NHS) and 10mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide salt with MES buffer Salt (EDC) solution;

[0125]Add 1mL NHS solution and 1mLEDC solution to polystyrene latex microspheres (100nm) solution in sequence, mix slowly at room temperature for 30 minutes, centrifuge at 19000g for 20 minutes after incubation, remove the supernatant, and use 10mL borax buffer (0.1 mol / L Na2B4O7, PH8.5) resuspension, shaking, ultrasonic treatment (ultrasonic breaker product model: YJ92-IIDN, power 50W, working time 2s, interval time 3s, alarm temperature 60℃, total time 30min), it becomes the activated latex Microspheres.

[0126]3.2)...

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Abstract

The invention relates to a preparation method of a latex immunochromatographic test strip for rapid detection of Klebsiella pneumoniae. The test strip is composed of a sample pad, a binding pad, a nitrocellulose membrane, a water-absorbing pad, and a PVC plate, and the binding pad is sprayed Anti-Klebsiella pneumoniae surface protein polyclonal antibody coupled to colored latex microspheres, detection line coated with anti-Klebsiella pneumoniae surface protein polyclonal antibody and goat anti-rabbit IgG antibody quality control Wire. When the added sample contains Klebsiella pneumoniae, Klebsiella pneumoniae first forms a complex with the latex-rabbit anti-Klebsiella pneumoniae surface protein polyclonal antibody, and migrates to the coated Klebsiella pneumoniae under capillary action. The detection line of the polyclonal antibody to the surface protein of Klebsiella pneumoniae is captured, and the detection line is colored accordingly, so that it can detect whether the sample contains Klebsiella pneumoniae. The test strip has the advantages of quickness, simplicity, high sensitivity and good specificity.

Description

Technical field[0001]The invention belongs to the field of biological detection, and specifically relates to a preparation method of latex microsphere immunochromatography test paper based on the surface protein of Klebsiella pneumoniae.Background technique[0002]Klebsiella pneumoniae (Klebsiella pneumoniae), also known as pneumonia bacillus or Friedlander bacillus, is the first gram-negative bacillus that can cause pneumonia. Half a century ago, gram-negative bacillary pneumonia (GNBP) was considered a very rare disease and received little clinical attention. Except for Klebsiella bacillus, there are almost no reports about gram-negative bacterium (GNB) causing pneumonia. In the past two to thirty years, with the changes in susceptible populations, the widespread use of antibacterial drugs, the changes in resistant bacteria, and the improvement and popularization of various microbial detection techniques, GNBP has become an important disease in modern medicine entering the era of an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C07K16/12C12N15/62C12N15/70G01N33/543G01N33/558G01N33/569G01N33/68
CPCC07K14/26C07K16/1203C07K2319/00C12N15/70G01N33/54313G01N33/558G01N33/56911G01N33/68
Inventor 杨波王猛胡征王毅
Owner HUBEI UNIV OF TECH