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Kit for accurately and quantitatively detecting vibrio vulnificus in food and detection method

A technology of vibrio vulnificus and kit, which is applied in the kit and detection field of quantitative detection of vibrio vulnificus, which can solve the problems of quantitative detection of vibrio vulnificus that have not been seen yet, and achieve shortened detection time, high tolerance and high accuracy degree and repeatability of the effect

Inactive Publication Date: 2017-12-15
BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU INSPECTION & QUARANTINE TECH CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the quantitative analysis of this technology for microorganisms is still in its infancy. Related studies such as human herpesvirus, methicillin-resistant Staphylococcus aureus, Chlamydia trachomatis, parasite Cryptosporidium in clinical samples, and Shigella in bovine feces samples Escherichia coli with toxins, and plant pathogenic bacteria that cause amylovora blight and potato brown rot, etc., have not yet seen relevant reports that can be used for the quantitative detection of Vibrio vulnificus in food

Method used

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  • Kit for accurately and quantitatively detecting vibrio vulnificus in food and detection method
  • Kit for accurately and quantitatively detecting vibrio vulnificus in food and detection method
  • Kit for accurately and quantitatively detecting vibrio vulnificus in food and detection method

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Experimental program
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Effect test

Embodiment 1

[0050] Embodiment 1 ddPCR primer, probe design

[0051] In order to achieve the specific detection and absolute quantitative analysis of Vibrio vulnificus in food, we selected the species-specific and highly conserved single-copy lipoprotein D gene (nplD, GenBank no.AY187681.1), RNA Polymerase subunit sigma factor S gene (rpoS, GenBank no.AY187681.1), virulence-related gene E (vcgE, GenBank no.AY626581.1) and metalloproteinase gene (met, GenBank no.U50548.1) as targets Sequences were analyzed and compared using NCBI online tools, and 4 pairs of primers and probe combinations were designed using Prime Express software V3.0 (ABI, Foster City, CA, USA). There are 4 sets of primer / probe combinations for ddPCR amplification conditions, and the sequences are shown in Table 1. Primers and probes were synthesized by Beijing Liuhetong Economic and Trade Co., Ltd. The primer / probe positions refer to GenBank no. AY187681.1 (nplD, rpoS gene), AY626581.1 (vcgE gene) and U50548.1 (met gen...

Embodiment 2

[0052] Example 2 Screening and specificity verification of primers and probes

[0053] Firstly, the designed and synthesized primers and probes were screened by qPCR method. Through the detection of the genomic DNA of Vibrio vulnificus (ATCC27562), it was found that all the primer probes could effectively amplify the target gene, but the fluorescent signal of the primer probe group met-F / R / P appeared earliest, which was compared with the negative control ratio, the fluorescence signal intensity is the largest, so the amplification effect is the best ( figure 1 A).

[0054] Next, in order to verify the specificity of the amplification reaction, the met-F / R / P primer probe was selected by qPCR method to detect the DNA of common Vibrio and food-borne pathogenic bacteria listed in Table 4. A total of 107 strains were used in this experiment (Table 4), including 1 standard strain of Vibrio vulnificus, 20 self-isolated strains, 66 other Vibrio standard / reference / self-isolated strai...

Embodiment 3

[0062] Embodiment 3 kit composition

[0063] The kit includes primers and probes for detecting Vibrio vulnificus designed in Example 1, a positive control (Vibrio vulnificus ATCC 27562 genomic DNA, 1000 copies / μL), a negative control (Escherichia coli ATCC 25922 genomic DNA, 1000 copies / μL), ddPCR master mix (purchased from BioRad, USA), microdroplet generation oil (purchased from BioRad, USA), microdroplet generation card (purchased from BioRad, USA), Twin Tec Semi-Skirted 96-well plate ( purchased from Eppendorf, Germany), and aluminum foil heat-sealing film (purchased from BioRad, USA).

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Abstract

The invention discloses a kit for accurately and quantitatively detecting vibrio vulnificus in food and a detection method and particularly relates to a group of primers and probes for detecting the vibrio vulnificus in food as well as the kit containing the primers and probes. The invention further provides a method for accurately and quantitatively detecting vibrio vulnificus in food by using a ddPCR (droplet digital PCR) technology. The method is independent of certificated standard substances or other standard substances, has higher accuracy, sensitivity and repeatability and is easy to standardize.

Description

technical field [0001] The invention belongs to the field of molecular biology bacteria detection, and more specifically relates to a kit and a detection method for quantitatively detecting Vibrio vulnificus. Background technique [0002] Vibrio vulnificus is a bacterium that commonly lives in the ocean. If raw seafood contaminated by this bacteria (especially oysters) is eaten, or skin wounds come into contact with shellfish or seawater where Vibrio vulnificus is present, infection can occur within 48 hours Sexual shock, skin and muscle necrosis, sepsis, and then cause multiple organ failure, the mortality rate is as high as 60%, which is the leading cause of death caused by seafood consumption in the United States. The current indicators for evaluating the safety of aquatic products still mainly rely on hygiene indicators such as coliform bacteria, which cannot reflect the contamination degree of Vibrio vulnificus. Although the existing regulations do not make clear regul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/06C12Q1/04C12N15/11C12R1/63
CPCC12Q1/686C12Q1/689C12Q2563/159C12Q2545/114
Inventor 魏海燕曾静马丹魏咏新徐蕾蕊李丹张西萌刘莉付溥博汪琦赵晓娟
Owner BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU INSPECTION & QUARANTINE TECH CENT