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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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The object of the present invention is to solve the problems existing in the detection method for th

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

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Example Embodiment

[0030] Example 1 Preparation of trivalent egg yolk antibody (IgY)

[0031] Take the Escherichia coli O157:H7, Listeria monocytogenes and Brucella strains stored at -80°C, and streak them on the LA plate after activation to separate individual colonies. After incubating at 37°C for 18-24 hours, pick a single colony. Inoculated in LB liquid medium and cultivated with shaking at 37°C to mid-log phase. Take 1 mL of the bacterial solution and use the plate pouring method to count the viable bacteria, add a formaldehyde solution with a final concentration of 1% to inactivate at room temperature for 30 minutes and overnight at 4°C. Centrifuge the inactivated bacterial solution at 3000 rpm for 10 minutes on the next day, discard the supernatant, collect the bacteria, resuspend the bacteria in saline to make a bacterial suspension, and adjust the final concentration of the three bacteria to 2×10 10 CFU·mL -1 , Placed in a refrigerator at 4℃ for later use. At the same time, the plate pour...

Example Embodiment

[0037] Example 2 Preparation of rabbit polyclonal antibody

[0038] Preparation of rabbit polyclonal antibodies against three zoonotic pathogens: Take the Escherichia coli O157:H7, Listeria monocytogenes and Brucella suspensions prepared in Example 1, respectively, and add an equal volume of Freund's complete adjuvant. Incomplete Freund’s adjuvant, place it on a magnetic stirrer and stir until it is completely emulsified, that is, water-in-oil state. After being placed at 4°C for a week, the emulsion will not delaminate. It can be used as a vaccine to immunize healthy female New Zealand white rabbits. After one week of adaptive feeding, there is no abnormality, and the immunization is carried out by subcutaneous injection of multiple points on the back. Freund's complete adjuvant vaccine was used for the first immunization, and then the same dose of Freund's incomplete adjuvant vaccine was used for booster immunization every 1 week. A total of 4 booster immunizations were used. E...

Example Embodiment

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

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

[0047] When E. coli O157:H7 is used as the coating antigen, the E. coli O157:H7 bacterial suspension is diluted 1:800 with the antigen coating solution, and 100μL per well is coated in a 96-well ELISA reaction plate and incubated at 37°C 1h, discard the liquid in the plate, add PBST buffer, wash 300μL / well, 3 min each time, wash 3 times; add 300μL 5% skimmed milk powder to each well, incubate at 37℃ for 2h, repeat the above washing steps; Test trivalent serum or antibody, 100μL / well, incubate at 37℃ for 0.5 h, repeat the above washing steps; Dilute the HRP-labeled goat anti-chicken secondary antibody at 1:5000, and add 100μL / well to the 96-well ELISA reaction plate Incubate...

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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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