Detection method for vibrio cholerae, vibrio parahaemolyticus and vibrio mimicus in foods

A technology for Vibrio cholerae and Vibrio hemolyticus, which is applied in the directions of microorganism-based methods, biochemical equipment and methods, and microbial determination/inspection, etc., can solve problems such as never seen before, and achieves reduction of detection costs and workload. , the effect of speeding up the detection speed

Inactive Publication Date: 2018-12-21
邵楚瑶
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  • Summary
  • 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 choler...

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Embodiment one: the detection of cuttlefish

[0015] 1. Aseptic operation, take 25g of breaded shrimp samples, cut them into pieces, add them into a sterile container filled with 225ml of 3% NaCl alkaline peptone water (APW), and incubate at 37°C±1°C for 4h to 15h.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 5. Centrifuge at 12000 rpm for 5 minutes, take the supernatant as a DNA template, and store it at -20°C for later use.

[0020] 6. Add sample and do PCR

[0021] 7. Take 10 μl of PCR reaction solution for agarose g...

Embodiment 2

[0023] Embodiment two: the detection of soft-shelled turtle eggs

[0024] 1. Aseptic operation, take 25g clam sample, cut it into pieces, put it into a sterile 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 PCR reaction solution for agarose gel electro...

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Abstract

The invention discloses a PCR (Polymerase Chain Reaction) detection method for vibrio cholerae, vibrio parahaemolyticus and vibrio mimicus in foods. The method is characterized by comprising the following steps: performing secondary enriched culture on a sample to be detected; extracting vibrio genome DNA (Deoxyribonucleic Acid) by adopting a water boiling method; then performing electrophoresis,dyeing and bleaching after a PCR reaction; observing the result with a gel imager and photographing; finally performing spectrum analysis to obtain a detection result. The detection method has the advantages that the vibrio cholerae, vibrio parahaemolyticus and vibrio mimicus in foods can be detected simultaneously, the detection speed can be improved under the condition of not influencing the detection rate, and the workload and detection cost are lowered.

Description

technical field [0001] The invention relates to a 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 design...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12R1/63
CPCC12Q1/686C12Q1/689C12Q2537/143Y02A50/30
Inventor 邵楚瑶刘滢佳陈晓旭
Owner 邵楚瑶
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