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Rapid detection method of microdroplet digital PCR for Vibrio mimicus in aquatic products

A mimetic Vibrio, droplet technology, applied in biochemical equipment and methods, microbial determination/inspection, resistance to vector-borne diseases, etc., to achieve the effect of reducing detection costs, fast detection, and high sensitivity

Active Publication Date: 2022-07-08
LINYI UNIVERSITY
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AI Technical Summary

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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Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0019] First, take 1 g of the sample to be tested aseptically, add it to a sterilized container containing 9 ml of 1% sodium chloride alkaline peptone water, and incubate it under aerobic conditions at 36 °C ± 1 °C for 24 hours. 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 the droplet digital PCR reaction. The total volume of each droplet digital PCR reaction is 25 μl, including 2 μl of the extracted genomic DNA; 12.5 μl of PCR master mix (2×ddPCR Master Mix); 1μl (10 pmol / μl), probe 0.5μl (10 pmol / μl); add ddH at the end 2 0 to 25 μl. Transfer the well-mixed PCR system to the droplet generating card, add 70 μl of special oil for droplet generating to the droplet generating card, and put the droplet generating card into the droplet generating card for reaction. All the generated drop...

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Abstract

The invention discloses a microdroplet digital PCR rapid detection method for mimetic vibrio in aquatic products. First, the genomic DNA of the enrichment solution of the aquatic product sample to be tested is extracted and diluted for use; then the conserved DNA sequence of the mimetic vibrio genome is used to design Specific primers and probes; then use the primers and probes to perform microdroplet digital PCR amplification and detection on the genomic DNA of the enriched bacteria solution of the aquatic product sample to be tested. After PCR amplification, the droplet analyzer was used to detect each droplet one by one. The droplet with fluorescent signal was interpreted as 1, and the droplet without fluorescent signal was interpreted as 0. Finally, according to the Poisson distribution principle and positive droplets The concentration or copy number of the target molecule to be detected is calculated. Droplet digital PCR can directly obtain the number of DNA molecules, and can perform absolute quantification of Vibrio mimicus in aquatic products. It 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 mimetic Vibrio ( Vibrio mimicus ) rapid detection method of droplet digital PCR. Background technique [0002] Foodborne disease is a public health problem that has attracted worldwide attention, and is currently the biggest food safety problem in China. Food pathogenic factors mainly include pathogenic microorganisms, natural toxins, parasites and toxic chemicals. At this stage, 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 pathogenic bacteria in microbial food poisoning incidents in my country, and it is of great significance to develop a rapid detection method for it. Vibrio mimetic is a gram-negative Vibrio with a single flagella, which is motile and exists in river water, seawater and aquatic products. Vibrio mimicus is widely distributed in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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