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Method for quantitative detection of pathogenic bacteria and total bacterial count in edible raw egg

A technology for quantitative detection and pathogenic bacteria, applied in the field of food hygiene, can solve the problems of difficult detection, high false positive rate, and low detection signal-to-noise ratio.

Pending Publication Date: 2019-09-10
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when the object of FCM detection is edible raw egg samples, there are the following problems: ①The viscous egg liquid cannot meet the requirements of FCM sampling, and the instrument injection tube is often blocked; ②The false positive rate is high; ③ Detection signal-to-noise ratio is low
Therefore, when the FCM detection object is edible raw egg samples, the detection is extremely difficult

Method used

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  • Method for quantitative detection of pathogenic bacteria and total bacterial count in edible raw egg
  • Method for quantitative detection of pathogenic bacteria and total bacterial count in edible raw egg
  • Method for quantitative detection of pathogenic bacteria and total bacterial count in edible raw egg

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] This example is used to illustrate the effectiveness of the pretreatment method for edible raw eggs and the influence of the pretreatment method on the detection of bacteria. The pretreatment experimental bacteria were obtained from laboratory culture.

[0084] The edible raw egg sample sampling plan used in this example is: put fresh eggs on a sterile ultra-clean bench, wipe the eggshells with 75% by volume ethanol, disinfect the eggshells, and then carefully break the eggshells, The egg content was placed in a 250mL sterile Erlenmeyer flask, shaken at 250rpm for 10min at 4°C, and a sterile egg liquid sample was prepared.

[0085] Take 0.1 mL of the sterile egg liquid sample prepared above, add 630 μL of PBS, 70 μL of proteinase K (20 mg / mL), and 200 μL of Triton X-100 (10 volume %), the total volume is 1 mL, that is, the egg liquid is diluted 10 times. The final concentration of the enzyme was 1.4 mg / mL, the final concentration of the degreasing reagent was 2% by volu...

Embodiment 2

[0090] This example is used to illustrate the comparison between the detection results of bacteria artificially added to edible raw eggs by the method provided by the present disclosure and the detection results of the traditional plate culture method.

[0091] In this experiment, the method of preparing egg liquid samples by eating raw egg samples is consistent with that in Example 1. Sampling of fresh eggs was carried out using the protocol in Example 1, and the bacterial culture method consistent with Example 1 was used to cultivate Escherichia coli K12.

[0092] The method for preparing the sterile egg liquid sample used in this example is: take 1 mL of the prepared Escherichia coli K12 bacterial liquid sample, centrifuge it and resuspend it in 1 mL of sterile raw egg sample, vortex and mix well, and obtain the sterile egg liquid sample .

[0093] Take 0.1 mL of the prepared sterile egg liquid sample, add 630 μL of PBS, 70 μL of proteinase K (20 mg / mL), and 200 μL of Trit...

Embodiment 3

[0098] This example is used to illustrate that the detection result of the ratio of target pathogenic bacteria and non-pathogenic bacteria artificially added to edible raw eggs by the method provided in the present disclosure is consistent with the actual addition ratio.

[0099] In this example, Alexa Fluor 488-labeled Salmonella typhimurium-specific monoclonal antibody probes were used for the first fluorescent labeling. The antibody was purchased from abcam with a product number of ab8247, and the antibody specifically recognized lipopolysaccharide (LPS) of Salmonella typhimurium. , Alexa Fluor 488 was purchased from Thermo Fisher, the product number is A2000, and the method of binding Alexa Fluor 488 to the antibody refers to the labeling strategy of Thermo Fisher, see Thermo Fisher A30006 method.

[0100] In this experiment, the method of preparing egg liquid samples by eating raw eggs is consistent with that in Example 1. The concentration of non-pathogenic bacteria mode...

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PUM

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Abstract

The invention relates to a method for quantitative detection of pathogenic bacteria and total bacterial count in an edible raw egg. The method comprises the following steps that S1, a to-be-detected edible raw egg sample is homogenized; S2, after an egg liquid sample is diluted, protein degradation treatment and degreasing treatment are carried out; S3, the treated egg liquid sample is subjected to membrane filtration; S4, the filtered egg liquid sample is subjected to centrifugal washing and supernatant removal, and the centrifuged precipitates are suspended by a suspending medium; S5, a specific pathogenic bacterium probe is used for performing first fluorescence labeling on an egg liquid suspension solution, and a nucleic acid dye is used for performing second fluorescence labeling on the egg liquid suspension solution; S6, the fluorescence of particles in a detection and sample loading suspension solution is detected by using high-sensitivity flow cytometry. The method is suitablefor detection of various edible raw egg and egg liquid samples, and can simultaneously and quantitatively detect target pathogenic bacteria and total bacterial count in the samples, including variouspathogenic bacteria, nonpathogenic bacteria and the like.

Description

technical field [0001] The present disclosure relates to the field of food hygiene, in particular to a method for quantitatively detecting pathogenic bacteria and total bacterial counts in edible raw eggs. Background technique [0002] Eggs have become an indispensable food for consumers because they contain rich nutrients such as protein, fat, and trace elements, and are low in price, high in bioavailability, and contain tryptophan, which is lacking in plant proteins. Become a "perfect food". [0003] However, eggs are susceptible to bacterial contamination during production, storage, and sales. Contaminating bacteria include two types: non-pathogenic bacteria and pathogenic bacteria. Eggs contaminated by non-pathogenic bacteria generally do not directly cause human diseases, but the nutritional value, processing value and storage time of eggs will be reduced. Therefore, the total number of bacteria in eggs has become a standard to measure the quality of eggs. Eggs conta...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/06G01N21/64
CPCC12Q1/06G01N21/6428G01N2021/6439Y02A50/30
Inventor 颜晓梅毛翠萍王旭正薛乘风
Owner XIAMEN UNIV
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