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Kit for synchronously and rapidly detecting various zoonotic pathogens

A technology for detecting kits and pathogenic bacteria, which is applied in the direction of measuring devices, instruments, scientific instruments, etc.

Inactive Publication Date: 2017-10-27
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The object of the present invention is to solve the problems existing in the detection method for the existing zoonotic pathogens, and provide a simultaneous rapid detection kit for various zoonotic pathogens

Method used

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  • Kit for synchronously and rapidly detecting various zoonotic pathogens
  • Kit for synchronously and rapidly detecting various zoonotic pathogens
  • Kit for synchronously and rapidly detecting various zoonotic pathogens

Examples

Experimental program
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Effect test

Embodiment 1 3

[0030] Example 1 Preparation of Trivalent Egg Yolk Antibody (IgY)

[0031] Take Escherichia coli O157:H7, Listeria monocytogenes and Brucella strains stored at -80°C, streak them on LA plates after activation, separate individual colonies, and pick individual colonies after culturing at 37°C for 18-24 hours. Inoculate in LB liquid medium and culture with shaking at 37°C until mid-logarithmic phase. Take 1 mL of the bacterial solution and use the plate pouring method to count viable bacteria, add formaldehyde solution with a final concentration of 1% to inactivate at room temperature for 30 min, and overnight at 4 °C. The next day, centrifuge the inactivated bacterial solution at 3000 rpm for 10 min, discard the supernatant, collect the bacterial cells, resuspend the bacterial cells with normal saline to make a bacterial suspension, and adjust the final concentrations of the three bacteria to 2×10 10 CFU mL -1 and stored in a 4°C refrigerator for later use. At the same time,...

Embodiment 2

[0037] Example 2 Preparation of Rabbit Polyclonal Antibody

[0038] Preparation of three kinds of zoonotic pathogenic bacteria rabbit polyclonal antibodies: respectively get Escherichia coli O157:H7 prepared in Example 1, Listeria monocytogenes and Brucella bacterial suspension, add an equal volume of Freund's complete adjuvant Adjuvant / Freund's incomplete adjuvant, put it on a magnetic stirrer and stir until it is completely emulsified, that is, the water-in-oil state. After standing at 4°C for a week, the emulsion does not separate, and it can be used as a vaccine to immunize healthy female New Zealand white rabbits , after a week of adaptive feeding, there was no abnormality, and the back subcutaneous multi-point injection method was used for immunization. Freund's complete adjuvant vaccine was used for the initial immunization, and then the same dose of Freund's incomplete adjuvant vaccine was used for booster immunization once every other week. There were 4 booster immuni...

Embodiment 3 3

[0045] Example 3 Trivalent egg yolk antibody (IgY) indirect ELISA method

[0046] In order to more accurately measure the titer of collected IgY, an indirect ELISA reaction method for detecting trivalent serum and egg yolk antibodies against three different pathogenic bacteria was established. The specific steps are:

[0047] When using Escherichia coli O157:H7 as the coated antigen, dilute the suspension of Escherichia coli O157:H7 bacteria 1:800 with the antigen coating solution, coat 100 μL per well in a 96-well enzyme-labeled reaction plate, and incubate at 37°C 1 h, discard the liquid in the plate, add PBST buffer, wash 300 μL / well, 3 min each time, wash 3 times; add 300 μL 5 % skimmed milk powder to each well, incubate at 37 ° C for 2 h, repeat the above washing steps; To measure trivalent serum or antibody, 100 μL / well, incubate at 37°C for 0.5 h, repeat the above washing steps; dilute the HRP-labeled goat anti-chicken secondary antibody at 1:5000, and add 100 μL / well i...

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Abstract

The invention discloses a kit for synchronously and rapidly detecting various zoonotic pathogens. The kit comprises immunomagnetic beads and a quantum dot fluorescent bioprobe; the immunomagnetic beads are obtained by activating carboxylated magnetic beads by EDC and NHS and coupling the beads with a trivalent yolk antibody IgY; the quantum dot fluorescent bioprobe is obtained by coupling fluorescent quantum dots with different colors with anti-escherichia coli O157: H7, anti-Listeria monocytogene, anti-Brucella and anti-vibrio parahaemolyticus rabbit polyclonal antibodies respectively; the trivalent yolk antibody IgY is obtained by deactivating escherichia coli O157: H7, Listeria monocytogene and brucella, and immunizing an SPF chicken. When the kit provided by the invention is used for performing synchronous rapid detection of four pathogens, the kit has the advantages of good specificity, high sensitivity, good reproducibility, good stability and short detection time; the kit can be applied to simultaneous rapid quantitative detection of the four pathogens.

Description

technical field [0001] The invention relates to the field of rapid detection, in particular to a simultaneous rapid detection kit for various zoonotic pathogenic bacteria. Background technique [0002] In recent years, with the rapid development of animal husbandry and tourism, the epidemiological characteristics of many zoonotic diseases have changed, with faster transmission and wider spread. Zoonotic infectious diseases not only seriously threaten the health of humans and animals, but also bring huge economic losses to agriculture and animal husbandry. Brucella, Escherichia coli O157:H7 and Listeria monocytogenes are three pathogenic bacteria with high incidence and serious harm to humans and animals among many zoonotic pathogens. Humans or animals often get sick by ingesting food contaminated by these bacteria. Establishing a simultaneous rapid and accurate detection method for these pathogens has become one of the research hotspots in the field of public health detecti...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/569
CPCG01N33/56911G01N33/56916G01N33/577G01N2333/23G01N2333/265
Inventor 李娟李丽宋丹丹宋秀玲徐坤王娟赵超刘玉申翟玥曲笑锋张惠雯郭媛媛李新新
Owner JILIN UNIV
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