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Kit for rapidly detecting staphylococcus aureus in milk

A Staphylococcus, aureus technology, applied in the direction of microorganism-based methods, microorganism measurement/inspection, microorganisms, etc., can solve the problems of poor sensitivity, cumbersome steps, and long time consumption, and achieve good specificity, good repeatability, Effects with simple steps

Inactive Publication Date: 2012-01-11
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the national standard method still adopts the traditional culture method, but the steps are cumbersome, time-consuming, high cost, poor sensitivity and other shortcomings
Although some simple and fast technologies have appeared in recent years, it is still not feasible to directly apply to the detection of Staphylococcus aureus in milk, because the protein and fat in milk have a great impact on the detection, so , the development of a rapid detection of Staphylococcus aureus in milk has become a major development trend

Method used

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  • Kit for rapidly detecting staphylococcus aureus in milk
  • Kit for rapidly detecting staphylococcus aureus in milk
  • Kit for rapidly detecting staphylococcus aureus in milk

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Composition and preparation of embodiment 1 kit

[0062] 1. Immunomicrospheres

[0063] 1) Preparation of polyclonal antibody against Staphylococcus aureus

[0064] A: Inoculate the identified Staphylococcus aureus into LB culture medium, cultivate overnight at 37°C on a shaker at 200r / min, count, inactivate with 0.3% formaldehyde solution at 37°C for 24 hours, and test for no viable bacteria on a plate with a physiological test. Wash twice with saline, and finally resuspend with 200 μL normal saline, and adjust its concentration to 1×10 9 CFU / ml, fully emulsified with 200 μL Freund's complete adjuvant, used as the immunogen.

[0065] B: Two male healthy New Zealand white rabbits weighing about 1 kg were taken, and the above immunogens were injected subcutaneously at multiple points. Two weeks after the first immunization, the booster immunization was given at a dose of 1×10 10 CFU / ml, 200 μL bacterial solution and 200 μL Freund’s incomplete adjuvant are fully emuls...

Embodiment 2

[0082] The specificity experiment of embodiment 2 kit

[0083] 1. Take Salmonella typhimurium, Shigella dysenteriae, Actinobacillus pleuropneumoniae serotype 5, Escherichia coli O157:H7, Yersinia enterocolitica, and Staphylococcus aureus that have been verified by DNA validity. 20 μL of the bacterial solution was placed in a centrifuge tube and heated at 95°C for 10 minutes for thermal lysis. Centrifuge at 12000rpm for 1min, and take the supernatant.

[0084] 2. Take 1 μL of the supernatants of various bacterial liquids and add them to the reaction system of the LAMP reaction solution for LAMP detection, and make negative and positive controls at the same time, and place them in a 65°C water bath for 50 minutes.

[0085] 3. Take 8 μL LAMP product, conduct electrophoresis detection with 2% agarose gel, and observe the amplification result on a gel image analyzer.

[0086] As a result, only Staphylococcus aureus was amplified, while Salmonella typhimurium, Shigella dysenteriae...

Embodiment 3

[0088] The sensitivity test of embodiment 3 kit

[0089] 1. Take 1 mL of overnight cultured Staphylococcus aureus culture solution and make a 10-fold dilution to 10 -9 . 20 μl of each dilution was placed in a PCR tube and heated at 95°C for 10 minutes for thermal lysis. Centrifuge at 12000rpm for 1min, and take the supernatant.

[0090] 2. Take 1 μL of the supernatant of each dilution and add it to the reaction system of the LAMP reaction solution for LAMP detection. At the same time, make negative and positive controls, and place it in a 65°C water bath for 50 minutes.

[0091] 3. Take 8 μL LAMP product, conduct electrophoresis detection with 2% agarose gel, and observe the amplification result on a gel image analyzer.

[0092] From the experimental results, it can be seen that when the bacterial solution is diluted to 10 -8 can still be amplified, the minimum detection limit is 50CFU / ml (see image 3 ). In the figure, 1 is the standard molecular weight of DL2000; 2 is ...

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Abstract

The invention discloses a method for rapidly detecting staphylococcus aureus in milk, belonging to the bioinstrumentation technical field and applicable to qualitative detection of staphylococcus aureus. The invention is composed of immune microballoon, two pairs of primers, DNA polymerase, LAMP reaction liquid, developing liquid and positive contrast liquid. The invention has rapid detection speed, good specificity, high sensibility, simple operation steps and good repeatability and can substitute the traditional method for detecting staphylococcus aureus in milk.

Description

technical field [0001] The invention discloses a kit for rapidly detecting staphylococcus aureus in milk, which uses a loop-mediated isothermal amplification (LAMP) technology to detect staphylococcus aureus in milk, and belongs to the technical field of biological detection. Background technique [0002] Staphylococcus aureus (S.aureus) is referred to as Staphylococcus aureus, spherical, with a diameter of 0.8 μm, arranged in clusters of grapes under the microscope, without spores, flagella, most of them without capsules, and Gram staining is positive. Staphylococcus aureus is ubiquitous in nature, and can be found in air, water, dust, and human and animal excrement. It is the main pathogen causing bacterial food poisoning, and it is listed as undetectable in the national fresh milk standard project. At present, the national standard method still adopts the traditional culture method, but the steps are cumbersome, time-consuming, high cost, poor sensitivity and other short...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12R1/445
Inventor 雷连成许会会孙长江谢芳杜崇涛冯新韩文瑜
Owner JILIN UNIV
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