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Rapid detection method based on magnetic separation and quantum dot labeling of Helicobacter pylori, and kit

A Helicobacter pylori, anti-Helicobacter pylori technology, applied in the direction of measuring devices, analysis materials, instruments, etc., can solve the problems of long pretreatment time, complex stool sample matrix, and can not meet the requirements of stool antigen detection, and achieve the detection method The effect of simplicity, low detection cost and high practical value

Pending Publication Date: 2019-11-05
GUANGDONG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The stool sample matrix is ​​complex and the pretreatment time is long. The traditional detection technology enzyme-linked immunosorbent assay (ELISA) cannot meet the requirements of stool antigen detection.

Method used

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  • Rapid detection method based on magnetic separation and quantum dot labeling of Helicobacter pylori, and kit
  • Rapid detection method based on magnetic separation and quantum dot labeling of Helicobacter pylori, and kit
  • Rapid detection method based on magnetic separation and quantum dot labeling of Helicobacter pylori, and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Preparation of Rabbit Anti-Helicobacter Pylori Outer Membrane Protein Polyclonal Antibody

[0065] 1. Preparation of rabbit anti-Helicobacter pylori outer membrane protein (OMPs) protein antigen

[0066] (1) Experimental method

[0067] 1. Culture of Helicobacter pylori (H.P)

[0068] H.P is a spiral, curved or straight unmycobacterium, 0.3~1.0×1.5~5μm, obtuse at the top, Gram negative, microaerophilic, and moves rapidly in a spiral form by 4 to 8 flagella at both ends. Use Columbia blood agar medium (add 5-10% of defibrated sheep blood) to culture colonies without obvious pigment, translucent, and small colonies in the form of needle points, put them into the incubator, adjust the culture conditions, and the gas conditions are 5% O 2 , 10% CO 2 and 85%N 2 (or H 2 ) in a gas environment with a temperature of 37° C. and a high-humidity environment for about three days to collect bacteria.

[0069] 2. Extraction of OMPs protein

[0070] The collected wet ...

Embodiment 2

[0078] Example 2 Preparation of Monoclonal Antibody Using the Urease B Peptide of Helicobacter pylori

[0079] 1. Experimental method

[0080] For the specific preparation process, refer to the method of the patent "Hybridoma Cell Line and Glycocholic Acid Monoclonal Antibody and Detection Kit Based on Hybridoma Cell Line" with application number 201810229091.4, using "Identification of an Antigenic Epitope in Helicobacter pylori Urease That Induces Neutralizing Antibody Production The monoclonal antibody was prepared from the urease B peptide CHHLDKSIKEDVQFADSRI of Helicobacter pylori provided in ".

[0081] The specific method is as follows:

[0082] (1) Animal immunity

[0083] The immunogen is hemocyanin coupled with peptide CHHLDKSIKEDVQFADSRI, which is directly dissolved in PBS to make a concentration of 1 mg / mL. Take 0.5mL of the above solution and emulsify it with an equal volume of complete Freund's adjuvant, inject it into Balb / C mice (5 6-week-old Balb / C female m...

Embodiment 3

[0101] Indirect immunofluorescence of embodiment 3pAb binding to bacteria

[0102] 1. Experimental method

[0103] (1) Fix: transfer the bacteria to a 1.5mL centrifuge tube (tip), centrifuge to remove the supernatant, add 500μL of ice-cold 4% paraformaldehyde, incubate at room temperature for 20min, and store in PBS at 4℃ for 1 week after fixation ;

[0104] (2) Permeabilization: Centrifuge to remove the supernatant, add 250 μL of permeabilization solution, and incubate at 4°C for 10 minutes;

[0105] (3) Glycine rinsing: centrifuge, absorb the permeate, rinse 3 times with glycine solution at room temperature;

[0106] (4) Blocking: After centrifuging and removing the glycine solution, add 250 μL of blocking solution and incubate at room temperature for 1.5 h;

[0107] (5) Primary antibody pAb incubation: centrifuge and remove the blocking solution, add 250 μL of the pAb of Example 1, and incubate at room temperature for 4 hours to overnight;

[0108] (6) Wash with PBST 3 ...

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Abstract

The invention discloses a rapid detection method based on magnetic separation and quantum dot labeling of Helicobacter pylori and a kit, and the kit comprises an anti-Helicobacter pylori immunomagnetic bead nano magnetic bead and an anti-Helicobacter pylori quantum dot-labeled nanoprobe. The invention utilizes the advantages that the immuno-nanomagnetic beads can enrich the sample, the separationspeed is fast, the efficiency is high and the like, and combines the characteristics of high photochemical stability and high fluorescence intensity of the quantum dots, so that the detection system has the effect of multi-signal synergistic amplification to have ultra-high detection sensitivity. The invention is simple in detection method, fast in detection, easy to determine and low in detectioncost, can perform early diagnosis and prevention with high coincidence rate of the clinical diagnosis, has very high practical value in clinical diagnosis, pathogen identification, epidemiological investigation, etc., and is easy to promote.

Description

technical field [0001] The invention relates to the technical field of Helicobacter pylori detection, more specifically, to a rapid detection method and kit for Helicobacter pylori based on magnetic separation and quantum dot labeling. Background technique [0002] Helicobacter pylori (H. pylori), a spiral-shaped Gram-negative bacterium, is a fastidious, slightly aerophilic human pathogen. In general, H. pylori infection is an acquired disease in early childhood, and in the absence of antibiotic treatment they remain infected throughout their lives. Currently, about 20% of the population in developing countries is affected, and more than 90% of the population in developing countries is also affected. [0003] It is unrealistic to conduct epidemiological analyzes or upper endoscopy in asymptomatic children and young adults with H. pylori infection. In addition, younger children are not suitable for serum antibody testing due to their lower sensitivity. 13 The C-urea breath...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N33/533G01N33/569
CPCG01N33/54326G01N33/533G01N33/56916
Inventor 赵肃清陈莉莉肖益热邹同达王甜甜
Owner GUANGDONG UNIV OF TECH
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