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A method for rapid screening of microbial strains producing fin algae toxins

A technology of fin algae toxins and microorganisms, applied in biochemical equipment and methods, measurement/inspection of microorganisms, etc., can solve problems such as heavy workload, high cost, and long cycle, and achieve low cost, short cycle, accuracy and The effect of high sensitivity

Active Publication Date: 2022-03-22
ZHEJIANG OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to overcome the problems of large workload, long period and high cost of the traditional screening method for toxin-producing strains, the present invention provides a method for rapidly screening microbial strains producing fin algae toxins by using DNA probe-colony in situ hybridization. Low speed, high speed, high accuracy and high sensitivity

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Sensitivity Test

[0028] (1) Pretreatment: Pick a single colony on the plate of Saccharospirillaceae sp. LZ-5 strain, extract its DNA sample according to the conventional method of molecular cloning, and dilute it 5 times; take 2uL of the diluted sample and spot it on nitrocellulose After soaking the filter membrane in 0.5M NaOH solution for 10 minutes, dry it naturally and place it in an oven at 80°C for 1.5 hours to fix the DNA; mL lysozyme; pH 7.5-8.0; put into DNA membrane, incubate at 37°C for 20 minutes, rinse with TE buffer to remove bacterial cell residues on the surface of DNA membrane;

[0029] (2) Place the DNA membrane pretreated in step (1) in 8-10mL hybridization buffer, the hybridization buffer is: 2×SSC solution, 50mM phosphate buffer, 1mM EDTA, 10% dextran sulfate, 50% deionized formamide; pH 7.0; incubate at 42°C for 45 minutes; place the labeled single-stranded DNA probe in boiling water for 8-12 minutes, put it in an ice bath, take 5 mL o...

Embodiment 2

[0032] Example 2 specificity test

[0033] (1) Pretreatment: 1 strain of toxin-producing bacteria (nitrate-reducing bacteria), 9 strains of non-toxin-producing bacteria (including Escherichia coli, Shigella sonii, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa , Vibrio harveii, Vibrio salmonicida, Moraxella lacunae, Pseudomonas shigellai), respectively pick single plate colonies of the above-mentioned sample strains, and extract their DNA samples according to the conventional method of molecular cloning. It is diluted 10 times;

[0034] Take 2uL of the diluted sample and spot it on a nitrocellulose filter, soak it in 0.5M NaOH solution for 10 minutes, then dry it naturally and put it in an oven at 80°C for 1.5 hours to fix DNA; add lysozyme to TE buffer, the amount of lysozyme added Add 6mg / mL lysozyme to each milliliter of TE buffer; pH is 8.0; put into DNA membrane, incubate at 38°C for 20 minutes, rinse with TE buffer to remove bacterial cell residues on ...

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Abstract

The invention relates to the technical field of marine biology, and in particular to a method for rapidly screening microbial strains producing fin algae, comprising the following steps: (1) pretreatment: making and fixing the DNA of the strain to be tested; (2) hybridization: mixing the strain to be tested The DNA of the strain is hybridized with the labeled single-stranded DNA probe; (3) Screening: Whether the strain to be tested produces finnolyxin is specifically detected. The invention can specifically detect whether the DNA sample of the strain to be tested produces pinotoxin through the in situ spot molecular hybridization of the colony, and the labeled single-stranded DNA probe has the characteristics of low cost, rapidity, high accuracy and sensitivity; low analysis cost, cycle time The reagents used are green and environmentally friendly, harmless to the human body, and have high market prospects and economic value.

Description

technical field [0001] The invention relates to the technical field of marine organisms, in particular to a method for rapidly screening microbial strains producing fin algae toxins. Background technique [0002] Dinophystoxins (DTXs) ​​are a type of diarrheal shellfish poisoning (DSP), which are highly toxic, fat-soluble, heat-stable polyether polyether marine biotoxins. DTXs are strong inhibitors of cell-specific protein phosphatases PP1 and PP2A, have long-term teratogenicity, and are a strong tumor promoter or primitive carcinogen. Its long-term toxic effect poses a serious threat to the marine ecological environment, shellfish farming and its export trade, and human life and health, and is a worldwide research hotspot. [0003] At present, fin algal toxins can be prepared by large-scale cultivation and product extraction of vortex dinoflagellates (such as dinoflagellates), but this method not only has a long cultivation period and high cost, but also requires special a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6841C12Q1/6834C12Q1/04
CPCC12Q1/6834C12Q1/6841C12Q2565/519C12Q2543/10C12Q2563/131
Inventor 张晓玲杨桥穆军蒋志伟张若男
Owner ZHEJIANG OCEAN UNIV
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