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Kit for detecting vibrio parahaemolyticus on basis of immunomagnetic beads and MnO2 nanometer particles

A hemolytic vibrio and nanoparticle technology, applied in the field of microbial detection, can solve the problems of low sensitivity, unfavorable sample on-site detection, pollution, etc.

Active Publication Date: 2017-09-26
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PCR technology can amplify the detection target in a short time and has the characteristics of high sensitivity, but the PCR operation process requires high requirements and is prone to false positives
The ELISA method uses antigen-antibody specific binding detection, which has low equipment requirements, but takes a long time and has relatively low sensitivity.
As an emerging detection technology, LAMP has the characteristics of low cost, high sensitivity, and high specificity. However, LAMP needs selective enrichment culture first, so it is not conducive to on-site detection of samples, and it may introduce new contamination

Method used

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  • Kit for detecting vibrio parahaemolyticus on basis of immunomagnetic beads and MnO2 nanometer particles
  • Kit for detecting vibrio parahaemolyticus on basis of immunomagnetic beads and MnO2 nanometer particles
  • Kit for detecting vibrio parahaemolyticus on basis of immunomagnetic beads and MnO2 nanometer particles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Preparation of Vibrio parahaemolyticus rabbit clone antibody IgG

[0024] Take the concentration as 1×10 9 CFU mL -1 The inactivated bacteria solution was emulsified completely with the same amount of Freund's complete adjuvant / Freund's incomplete adjuvant to prepare the inactivated vaccine. The concentration for the first immunization was 1×10 9 CFU mL -1 Healthy New Zealand white rabbits were immunized with Freund's complete adjuvant vaccine (provided by the Experimental Animal Center of the Basic Medical College of Jilin University). Rabbits were immunized with the incomplete adjuvant vaccine for the second time and the third time. After 10 days, the immunization was boosted twice with inactivated bacterial solution. Before each immunization, blood was collected from the vein of the rabbit's ear, and the serum titer was determined. When the separation standard is reached, blood is collected from the heart of the rabbit, and the serum is separated to...

Embodiment 2

[0027] Example 2 Preparation of Vibrio parahaemolyticus chicken yolk antibody IgY

[0028] Take the concentration as 1×10 9 CFU mL -1 The inactivated bacterial solution was emulsified completely with the same amount of Freund's complete adjuvant / Freund's incomplete adjuvant to prepare the inactivated vaccine, which was used to immunize laying hens. The vaccine was inoculated into chicken breasts by intramuscular multi-point injection. Freund's complete adjuvant vaccine was used for the first immunization, 1 mL per chicken, and the Freund's incomplete adjuvant vaccine was used for the second immunization two weeks later, followed by a booster immunization every two weeks. Eggs before and after immunization were collected and stored in a 4°C refrigerator for later use. The yolk antibody in eggs was extracted by PEG 6000 salting-out method. The titer and specificity of yolk antibody before and after purification were measured by indirect ELISA method. Table 3 is the chicken ...

Embodiment 3

[0031] Example 3 Preparation of Immunomagnetic Beads

[0032]Add 1.08g FeCl 3 .6H 2 O into 22mL ethylene glycol, ultrasonically dissolved for 10min to form a reddish-brown solution; then add 1.2g NaAc and 0.2g sodium citrate to the solution, ultrasonically for 10min to form a dark red solution; finally, add 0.2gPEG6000 to the above solution , ultrasonically mixed, the resulting homogeneous solution was transferred to a reaction kettle, and reacted in an oven at 199°C for 18h. Use a permanent magnet to separate the medium-black solid matter from the reaction solution, wash it alternately with ultrapure water and ethanol for 3-5 times, and obtain Fe 3 o 4 Nanoparticles; take 0.5 mg carboxylated Fe 3 o 4 Nanoparticles, washed twice with PBS, resuspended in 1mL PBS, added 10mg EDC and 10mg NHS to activate the carboxyl group, after 30min, magnetically adsorbed, removed the supernatant, washed 3 times with PBS, resuspended in 1mL PBS, added 50µg anti-parahemolytic Vibrio rabbi...

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Abstract

The invention discloses a kit for detecting vibrio parahaemolyticus on the basis of immunomagnetic beads and MnO2 nanometer particles. Magnetic beads with superparamagnetism are prepared; the magnetic beads are coupled with vibrio parahaemolyticus polyclonal antibodies; MnO2 nanometer particles are synthesized; the nanometer particles are coupled with vibrio parahaemolyticus specific chicken egg-yolk antibodies; liquid to be tested is taken and is mixed with two kinds of probes; a magnetic force frame is used for separating magnetic beads-thallus-MnO2 compounds; a citrate buffer solution is used for resuspending the mixture; TMB is added for color development, so that the fast specific detection of the vibrio parahaemolyticus is realized. The vibrio parahaemolyticus detection by using the immunomagnetic beads and MnO2 nanometer particle technology is provided by the invention; the immunological reaction of an ELISA method is used for color development; the detection time is shortened; during the quantitative detection, the variation coefficient is small; the lowest detection concentration is 10 CFU / mL; the adding standard recovery rate reaches 96.7 percent; the sensitivity is high; the stability is high.

Description

technical field [0001] The invention belongs to the field of microbial detection, in particular to a method based on immunomagnetic beads and MnO 2 Nanoparticle detection kit for Vibrio parahaemolyticus. Background technique [0002] Vibrio parahaemolyticus ( Vibrio parahaemolyticus , VP) is a Gram-negative non-spore-forming halophile, which is one of the common foodborne pathogens. The bacterium is mainly derived from seafood such as shrimp, fish and shellfish, and mainly causes acute gastroenteritis, wound infection and sepsis in humans through contaminated seafood. In recent years, with the continuous expansion of the population of eating seafood, the food poisoning caused by Vibrio parahaemolyticus is on the rise. According to reports, since 1998, the food poisoning caused by Vibrio parahaemolyticus has exceeded that of Salmonella. bacteria food poisoning, becoming the most serious foodborne pathogens. At present, this bacterium is a pathogenic bacterium that must be...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/543
CPCG01N33/54326G01N33/54346G01N33/56911
Inventor 赵超刘玉申李娟王娟付凯悦聂玮宋秀玲徐坤
Owner JILIN UNIV
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