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Method for detecting food-derived pathogenic vibrio bacteria by composite fluorescence PCR technique

A pathogenic Vibrio and compound fluorescence technology, applied in the detection of pathogenic Vibrio and food-borne pathogenic Vibrio, can solve the problems of difficult biochemical identification of molecular biology detection methods, save detection cost and time, High sensitivity and time saving effect

Inactive Publication Date: 2008-01-30
ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the development of molecular biology, the bacterial identification methods in food inspection and quarantine work have developed from the traditional simple biochemical experiment level to molecular biological methods such as PCR, probe hybridization and other technologies. In the short term, it is still difficult to replace traditional biochemical identification. Currently, molecular biology methods are mainly used for primary screening of a large number of samples.

Method used

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  • Method for detecting food-derived pathogenic vibrio bacteria by composite fluorescence PCR technique
  • Method for detecting food-derived pathogenic vibrio bacteria by composite fluorescence PCR technique
  • Method for detecting food-derived pathogenic vibrio bacteria by composite fluorescence PCR technique

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Sample: hairtail exported somewhere.

[0050] Use conventional physiological and biochemical methods to detect suspected colonies of Vibrio parahaemolyticus, and then perform the following composite fluorescent PCR technology to detect foodborne pathogens:

[0051] 1. Sample Processing

[0052] (1) Take 100 grams of the sample to be tested and pulverize it.

[0053] (2) Dissolve in 1 liter of nutrient broth and incubate at 37°C for 8 hours.

[0054] 2. DNA extraction

[0055] Take 1 mL of nutrient broth and let it stand in an ice bath for 5 minutes, then centrifuge at 12,000 rpm for 5 minutes at room temperature, discard the supernatant, add 100 μL of lysozyme solution, incubate at 37°C for 10 minutes, and add 500 μL of TE buffer , shake to mix. Add the same volume of Tris-saturated phenol (pH 8.0), shake vigorously, centrifuge at 12,000 rpm for 3 minutes, take the supernatant, and repeat the phenol extraction. Take the supernatant, add 0.1 times the volume of sodi...

Embodiment 2

[0084] Sample: Somewhere fish balls are exported.

[0085] Use conventional physiological and biochemical methods to detect suspected colonies of Vibrio vulnificus, and then perform the following composite fluorescent PCR technology to detect foodborne pathogenic Vibrio:

[0086] 1. Sample Processing

[0087] (1) Take 100 grams of the sample to be tested and pulverize it.

[0088] (2) Dissolve in 1 liter of nutrient broth and incubate at 37°C for 8 hours.

[0089] 2. DNA extraction

[0090] Take 1 mL of nutrient broth and let it stand in an ice bath for 5 minutes, then centrifuge at 12,000 rpm for 5 minutes at room temperature, discard the supernatant, add 100 μL of lysozyme solution, incubate at 37°C for 10 minutes, and add 500 μL of TE buffer , shake to mix. Add the same volume of Tris saturated phenol (pH 8.0), shake vigorously, centrifuge at 12,000 rpm for 3 minutes, take the supernatant, and repeat the phenol extraction. Take the supernatant, add 0.1 times the volume...

Embodiment 3

[0119] Sample: Somewhere export kelp.

[0120] Use conventional physiological and biochemical methods to detect suspected colonies of Vibrio cholerae, and then perform the following composite fluorescent PCR technology to detect foodborne pathogenic bacteria:

[0121] 1. Sample Processing

[0122] (1) Take 100 grams of the sample to be tested and pulverize it.

[0123] (2) Dissolve in 1 liter of nutrient broth and incubate at 37°C for 8 hours.

[0124] 2. DNA extraction

[0125] Take 1 mL of nutrient broth and let it stand in an ice bath for 5 minutes, then centrifuge at 12,000 rpm for 5 minutes at room temperature, discard the supernatant, add 100 μL of lysozyme solution, incubate at 37°C for 10 minutes, and add 500 μL of TE buffer , shake to mix. Add the same volume of Tris-saturated phenol (pH 8.0), shake vigorously, centrifuge at 12,000 rpm for 3 minutes, take the supernatant, and repeat the phenol extraction. Take the supernatant, add 0.1 times the volume of sodi...

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Abstract

The invention discloses a method by using composite fluorescence PCR technique to detect food-borne pathogenic vibrio and pertains to bacteria detection technical field. The main technic proposal is to design a primer group sequence. The pathogenic vibrio is common pathogenic bacteria in food and has a serious effect on human health. The quick and accurate detection of pathogenic vibrio in food is a main premise condition for effective prevention and control of pathogen bacteria infection. The food-borne pathogenic vibrio required detection mainly comprises parahemolytic vibrio, vibrio cholerae and vibrio vulnificus. The invention overcomes technical shortage in the prior art aiming at the object bacteria and provides the quick and low-cost detection method by using composite fluorescence PCR technique to detect parahemolytic vibrio, vibrio cholerae and vibrio vulnificus. The method can use one-diode PCR reaction and primarily screen parahemolytic vibrio, vibrio cholerae and vibrio vulnificus.

Description

technical field [0001] The invention relates to a bacterium inspection technology, specifically a technology for detecting pathogenic vibrio, especially food-borne pathogenic vibrio, by using fluorescent PCR reaction. Background technique [0002] Common pathogenic bacteria in food are one of the main causes of food poisoning, which can lead to a variety of diseases and seriously threaten people's health. Among them, Vibrio is a more serious food-borne bacteria. Rapid and accurate detection of pathogenic bacteria in food is a prerequisite for effective prevention and control of pathogenic bacteria infection. With the development of molecular biology, the bacterial identification methods in food inspection and quarantine work have developed from the traditional simple biochemical experiment level to molecular biological methods such as PCR, probe hybridization and other technologies. In the short term, it is still difficult to replace traditional biochemical identification. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCY02A50/30
Inventor 郑文杰黄熙泰叶露萌张宏伟唐丹舟于佳李永君魏亚东刘寅
Owner ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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