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Micro-droplet type digital PCR rapid detection method for vibrio mimicus in aquatic products

A technology for mimicking Vibrio and detection methods, applied in biochemical equipment and methods, microbial determination/inspection, resistance to vector-borne diseases, etc., to achieve the effects of reducing detection costs, high sensitivity, and improving sensitivity

Active Publication Date: 2020-01-10
LINYI UNIVERSITY
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Problems solved by technology

However, so far, there have been no reports in China that are conducive to the rapid detection of Vibrio mimicus by droplet digital PCR method

Method used

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Embodiment Construction

[0019] First, take 1g of the sample to be tested aseptically, add it to a sterile container filled with 9ml of 1% sodium chloride alkaline peptone water, and incubate at 36°C±1°C for 24h under aerobic conditions. The cultured bacterial solution was added to a centrifuge tube, centrifuged at 12,000 rpm for 1 min, the supernatant was discarded, and genomic DNA was extracted by bacterial precipitation and diluted for later use.

[0020] Then carry out micro-droplet digital PCR reaction, the total volume of each micro-droplet digital PCR reaction is 25 μl, including 2 μl of extracted genomic DNA; PCR master mix (2×ddPCR Master Mix) 12.5 μl; 1μl (10 pmol / μl), probe 0.5μl (10 pmol / μl); finally add ddH 2 0 to 25 μl. Transfer the well-mixed PCR system to the droplet generator card, add 70 μl of special oil for droplet generator to the droplet generator card, and put the droplet generator card into the droplet generator for reaction. All the generated micro-droplets were transferred ...

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Abstract

The invention discloses a micro-droplet type digital PCR rapid detection method for vibrio mimicus in aquatic products. The micro-droplet type digital PCR rapid detection method for the vibrio mimicusin the aquatic products comprises the steps that genome DNA of enrichment broth of an aquatic product sample to be detected is first extracted and diluted for standby application; then the conservative DNA sequence design of a vibrio mimicus genome is used for designing specific primers and probes; and then the primers and the probes are used for performing micro-droplet type digital PCR amplification and detection on the genome DNA of the enrichment broth of the aquatic product sample to be detected. After PCR amplification, each micro-droplet is detected one by one using a micro-droplet analyzer, a micro-droplet with a fluorescent signal is interpreted as 1, the micro-droplet without the fluorescent signal is interpreted as 0, finally, the concentration or copy number of a target molecule to be detected is calculated according to the Poisson distribution principle and the proportion of positive micro-droplets, the micro-droplet type digital PCR can directly obtain the number of DNAmolecules, and absolute quantification can be performed on the vibrio mimicus in the aquatic products. The micro-droplet type digital PCR rapid detection method for the vibrio mimicus in the aquatic products has the advantages of high sensitivity, accurate quantification, wide linear range of detection, good specificity, and moderate cost.

Description

technical field [0001] The present invention relates to a kind of Vibrio mimicus ( Vibrio mimicus ) droplet digital PCR rapid detection method. Background technique [0002] Foodborne disease is a public health issue of global concern, and it is currently the biggest food safety issue in China. Food pathogenic factors mainly include pathogenic microorganisms, natural toxins, parasites and toxic chemicals. At present, the main cause of unqualified food and food poisoning is microbial contamination, and it is very important to prevent and control the contamination of pathogenic microorganisms. Vibrio mimicus is an important pathogen in microbial food poisoning incidents in my country, and it is meaningful to develop its rapid detection method. Mimic Vibrio is a Gram-negative Vibrio with a single flagella and power, present in river water, sea water and aquatic products. Mimic Vibrio is widely distributed in Fujian, Jiangsu, Zhejiang, Shanghai and other coastal areas of Chi...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/06
CPCC12Q1/689C12Q1/6851C12Q2563/159C12Q2531/113C12Q2563/107Y02A50/30
Inventor 刘云国韩庆典段家玉康大成彭善丽
Owner LINYI UNIVERSITY
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