Method for rapidly screening microorganism strain capable of generating gonyautoxin and digoxin labeled DNA (Deoxyribonucleic Acid) probe used by method

A technology for labeling of gonitoxin and digoxin, which is applied in the field of marine biology, can solve the problems of rapid screening of bacterial strains, heavy workload, long cycle, etc., and achieve the effect of not easy to operate errors and low errors

Active Publication Date: 2018-12-11
ZHEJIANG OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In order to solve the current inability to quickly screen strains capable of producing GTXs, it can only be achieved through chemical analysis of metabolites, which not only has a large workload and a long cycle, but also the toxin standards used in chemical analysis are expensive and costly to analyze. Higher problem, the present invention provides a kind of rapid screening method that produces gonitoxin microbial strain, can quickly screen a large number of mixed bacterial strains by probe labeling

Method used

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  • Method for rapidly screening microorganism strain capable of generating gonyautoxin and digoxin labeled DNA (Deoxyribonucleic Acid) probe used by method

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Sensitivity test sample strain cultivation, take samples of nitrate-reducing bacteria (Nitratireductor sp.LZ3-1, chemical analysis of its metabolites confirmed to produce GTXs toxin), pick a single colony of the LZ3-1 strain on the plate, and extract its For DNA samples, dilute it 5-10 times.

Embodiment 2

[0031]Specific detection sample strain cultivation, take samples of 1 strain of toxin-producing bacteria (nitrate-reducing bacteria), 9 strains of non-toxin-producing bacteria (including Escherichia coli, Shigella sonneri, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa bacterium, Vibrio harveii, Vibrio salmonicida, Moraxella lacunae, Pseudomonas shigellai), pick single colonies on the plate of each strain, and extract their DNA samples according to the conventional method of molecular cloning, Dilute it 5-10 times.

Embodiment 3

[0033] A method for rapid screening of microorganism strains producing gonitoxins, said method comprising the following steps:

[0034] 1) Take 2 μL of the sample prepared in Example 1 or Example 2 and put it on a nitrocellulose membrane to make the membrane to be tested, soak the membrane to be tested in 0.3mol / L NaOH solution for 5min, and place it at 60°C after immersion Dry for 1 hour to immobilize DNA, then place it in 3 mg / mL lysozyme solution with pH 7.6, warm water bath at 35°C for 15 minutes, wash off bacterial cell residues on the membrane surface with TE buffer to obtain pretreated DNA membrane , placing the obtained pre-treated DNA membrane in a hybridization solution, and incubating at 40°C for 40 minutes to obtain a pre-hybridization solution;

[0035] 2) Put the digoxin-labeled DNA in boiling water for 8 minutes and cool it in an ice bath to obtain a labeled DNA solution. Take the labeled DNA solution and add it to the pre-hybridization solution obtained in step...

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Abstract

The invention relates to the technical field of marine organisms and in particular relates to a method for rapidly screening a microorganism strain capable of generating gonyautoxin and a digoxin labeled DNA (Deoxyribonucleic Acid) probe used by the method. According to the method provided by the invention, the utilized probe is single-stranded DNA with a length of 248 basic groups which are complementary with a DNA basic group to be detected, and a 3' terminal of the probe is labeled by digoxin. Colony in-situ dot blot molecular hybridization is carried out, and the probe can be used for specifically detecting whether a strain DNA sample to be detected has tetrodotoxin synthesizing (sxtS) gene nucleic acid or not, and a toxin-producing positive strain is screened. According to the methodprovided by the invention, DNA in-situ extract of the strain is used for carrying out colony dot hybridization, and 96 samples can be screened within 8 to 10 hours. The method provided by the invention is rapid, high in specificity and high in accuracy.

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 gonitoxins and a digoxin-labeled DNA probe used therein. Background technique [0002] Gonyautoxins (GTXs) are one of the paralytic shellfish toxins, which belong to guanamine neurotoxins and are specific sodium ion channel blockers. GTXs have important applications in the detection of harmful red tides, neurophysiology, medical diagnosis, drug development, biochemical warfare agents and other research. In medical diagnosis and drug development, because of its unique chemical structure and toxicological mechanism, it has become + An important tool medicine for channels. And it has efficient analgesic, anesthesia, antispasmodic, antihypertensive, antiasthmatic and other functions, and it is an important lead compound for the development of new marine drugs. Some marine dinoflagellates, freshwater cyanobacteria ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6841C12Q1/6834C12Q1/04C12N15/11
CPCC12Q1/6834C12Q1/6841C12Q2543/10C12Q2563/131
Inventor 张晓玲杨桥穆军蒋志伟张若男
Owner ZHEJIANG OCEAN UNIV
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