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PCR detection method for vibrio cholerae, vibrio parahaemolyticus, and vibrio mimicus in food

A technology for Vibrio cholerae and Vibrio hemolyticus, which is applied in biochemical equipment and methods, microbial determination/inspection, measurement devices, etc., can solve problems such as never seen before, and achieves reduction of detection cost, acceleration of detection speed, and reduction of The effect of workload

Inactive Publication Date: 2007-05-23
INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When domestic inspectors design PCR detection methods, they also use the virulence gene as the sequence to be amplified, and most of the attention is on O1 and O139 Vibrio cholerae and Vibrio parahaemolyticus. The report on the detection of Vibrio vulnificus by PCR method was only in 2000 There has been 1 case of Vibrio mimicus, which has not been seen so far

Method used

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  • PCR detection method for vibrio cholerae, vibrio parahaemolyticus, and vibrio mimicus in food
  • PCR detection method for vibrio cholerae, vibrio parahaemolyticus, and vibrio mimicus in food
  • PCR detection method for vibrio cholerae, vibrio parahaemolyticus, and vibrio mimicus in food

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Experimental program
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Effect test

Embodiment 1

[0023] Embodiment one: the detection of cuttlefish

[0024] 1. Aseptic operation, take 25g breaded shrimp sample, cut it into pieces, put it into a sterilized container filled with 225ml 3% NaCl alkaline peptone water (APW), and incubate at 37°C±1°C for 4h-15h.

[0025] 2. Take 1ml of the enrichment solution and add it to a sterilized test tube filled with 9ml of 3% NaCl alkaline peptone water (APW), and incubate at 37°C±1°C for 4h to 15h.

[0026] 3. Pipette gun to absorb 1.5ml of the cultured bacterial solution, add it to a 1.5ml centrifuge tube and centrifuge at 13000rpm for 1min, discard the supernatant.

[0027] 4. Add 0.6ml of sterile deionized water to suspend the precipitate, shake and mix well, and bathe in 100°C water bath for 10min.

[0028] 5. Centrifuge at 12000 rpm for 5 minutes, take the supernatant as a DNA template, and store it at -20°C for later use.

[0029] 6. Add sample and do PCR

[0030] 7. Take 10 μl of the PCR reaction solution for agarose gel elec...

Embodiment 2

[0033] Embodiment two: the detection of soft-shelled turtle eggs

[0034] 1. Aseptic operation, take 25g clam sample, cut it into pieces, put it into a sterilized container filled with 225ml 3% NaCl alkaline peptone water (APW), and incubate at 37°C±1°C for 4h-15h.

[0035] 2. Take 1ml of the enrichment solution and add it to a sterilized test tube filled with 9ml of 3% NaCl alkaline peptone water (APW), and incubate at 37°C±1°C for 4h to 15h.

[0036] 3. Pipette gun to absorb 1.5ml of the cultured bacterial solution, add it to a 1.5ml centrifuge tube and centrifuge at 13000rpm for 1min, discard the supernatant.

[0037] 4. Add 0.6ml of sterile deionized water to suspend the precipitate, shake and mix well, and bathe in 100°C water bath for 10min.

[0038] 5. Centrifuge at 12000 rpm for 5 minutes, take the supernatant as a DNA template, and store it at -20°C for later use.

[0039] 6. Add sample and do PCR

[0040] 7. Take 10 μl of the PCR reaction solution for agarose gel ...

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Abstract

The PCR detection method for the cholerae vibrio, parahaemolyticus vibrio and mimicus vibrio in food, the characteristic is: its detection steps: after the detected sample being secondary enrichment, using boiled method to extract vibrio genomic DNA, and then after PCR reaction, electrophoresis, dyeing, washing, finally using gel imager to observe the result and photograph, and after the spectra analysis, the results can be derived. The invention has the advantage: the simultaneous detection of cholerae vibrio, parahaemolyticus vibrio and mimicus vibrio in food, and can accelerate speed under the conditions of not affecting the detection rate, and can reduce workload and detection cost.

Description

technical field [0001] The invention relates to a PCR detection method for vibrio cholerae, vibrio parahaemolyticus and vibrio mimicus in food. Background technique [0002] At present, domestic detection methods for pathogenic Vibrio are still traditional microbiological detection methods, which are not perfect enough. Each detection method can only detect one type of pathogenic Vibrio, while foreign traditional microbiological detection methods (such as FDA , AOAO, ISO, etc.) are also programs designed for a single or a small number of target bacteria. Therefore, almost every detection of a pathogenic Vibrio requires the preparation of different culture media and reagents, which is time-consuming and labor-intensive. In terms of molecular biology detection methods, the FDA2004 standard is still to design a PCR program for the purpose of detecting a pathogenic Vibrio, and the detection of different Vibrio requires different reaction conditions. When domestic inspectors de...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/447G01N21/84C12Q1/68
CPCY02A50/30
Inventor 黄晓蓉郑晶陈彬吕海沧
Owner INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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