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Kit for rapid detection of staphylococcus aureus in sample and detection method thereof

A staphylococcus, golden yellow technology, applied in the field of immunological detection, can solve the problems of long time consumption, low detection sensitivity, complicated and cumbersome inspection procedures, etc., and achieve the effect of fast and accurate detection.

Inactive Publication Date: 2012-01-18
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Traditional methods for detecting bacteria include biochemical culture, isolation and identification, the inspection procedures are complex and cumbersome, and it takes about 4-7 days to report the inspection results, which takes too long and the detection sensitivity is low

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1. Immunogen and positive standard preparation

[0050] Staphylococcus aureus (ATCC 29213) was inoculated on a Baird-Parker plate, cultured at 37°C for 24h, picked a single colony in 10% sodium chloride tryptone soybean broth at 37°C, 200r / min shaking culture for 17h, counted, and used 1% formaldehyde was inactivated overnight at 4°C, and the concentration of normal saline was adjusted to 5×10 9 cfu / mL, for the immunogen; adjust the concentration to 10 with PBS buffer 7 cfu / mL is the positive control standard, and PBS buffer is the negative control standard.

Embodiment 2

[0051] Embodiment 2. Preparation of coating antiserum

[0052] 1) Experimental animals: Three healthy New Zealand white rabbits aged 2 months and weighing 1.5-2 kg were selected as experimental animals.

[0053] 2) Emulsification: Emulsify 0.5mL immunogen with an equal amount of complete or incomplete Freund's adjuvant by mixing and stirring until a drop of emulsion is dropped into the water and floats on the water surface without dispersing.

[0054] 3) Immunization method: For the first immunization, inject the emulsified reagent at multiple points on the back of the rabbit, 1 mL / rat. The immunization after the initial immunization is called booster immunization, and the booster immunization is emulsified with Freund's incomplete adjuvant.

[0055] 4) Blood collection: After 3 booster immunizations, blood was collected from the ear vein, and the antiserum titer was determined by indirect non-competitive enzyme-linked immunoassay. After the titer reached the requirement (1...

Embodiment 3

[0057] Embodiment 3. Preparation of monoclonal antisera

[0058] 1) Experimental animals: 3 8-week-old female BALB / C mice weighing about 20 g were selected as experimental animals.

[0059] 2) Immunization method: each mouse was intraperitoneally injected with 0.2 mL of immunogen, and the same dose was boosted once every two weeks.

[0060] 3) Blood collection: Blood was collected from the tail vein after 3 booster immunizations, and the antiserum titer was determined by indirect non-competitive ELISA. When the titer no longer rises, the same amount of immunogen is injected intraperitoneally, and cell fusion is carried out according to the conventional method 3 days later.

[0061] 4) Cell fusion: the spleen cells of the immunized mice were conventionally fused with SP2 / 0 myeloma cells under the action of 50% PEG, respectively inoculated in 96-well culture plates, and placed in 37°C, 5% CO 2 Incubator cultivation.

[0062] 5) Screening of hybridoma cells: using indirect no...

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Abstract

The invention discloses a kit for rapid detection of staphylococcus aureus in a sample and a detection method thereof, belonging to the technical field of immunological detection. In the invention, a staphylococcus aureus immunogen inactivated with formaldehyde is used for immunizing a healthy New Zealand rabbit to obtain a polyclonal antibody to serve as a coated antibody, and used for immunizing a BALB / C mouse and performing cell fusion to obtain a monoclonal antibody to serve as a secondary antibody, thus, the kit for performing a double antibody sandwich enzyme-linked immunosorbent assay on the staphylococcus aureus in food (milk) is established, and a rapid and efficient detection means is provided for residual detection of the staphylococcus aureus in the food, and the advantages of lower cost and better stability and repeatability are achieved. A detection limit of the kit is 105cfu / mL and is suitable for detecting mass samples.

Description

technical field [0001] A kit and a detection method for rapidly detecting Staphylococcus aureus in a sample belong to the technical field of immunological detection. Background technique [0002] Staphylococcus aureus is an important pathogenic bacterium of zoonosis, which widely exists in air, water, dust and human and animal excreta in nature. Both humans and animals have a high rate of bacteria transmission. Healthy people often have enterotoxin-producing (SE) strains in their throats, nasal cavities, skin, and hair, and there are many chances of food being contaminated by them. In recent years, the US Centers for Disease Control reported that infections caused by Staphylococcus aureus accounted for the second place, second only to Escherichia coli. Staphylococcus aureus enterotoxin is a worldwide health problem. In the United States, food poisoning caused by Staphylococcus aureus enterotoxin accounts for 33% of the total bacterial food poisoning, and Canada has more, ac...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/569G01N21/31
Inventor 王利兵胥传来勇倩倩邓小芳宋珊珊张勋
Owner JIANGNAN UNIV
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