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A method for rapidly screening microbial strains producing tetrodotoxin and the used digoxin-labeled dna probe

A technology of digoxin labeling and DNA probes, which is applied in biochemical equipment and methods, microbial measurement/inspection, recombinant DNA technology, etc., can solve the problems of heavy workload, high analysis cost, and long cycle, and achieve unlimited Easy operation error, high efficiency, high precision effect

Active Publication Date: 2022-03-22
ZHEJIANG OCEAN UNIV
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  • Abstract
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  • Claims
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Problems solved by technology

[0004] In order to solve the current inability to quickly screen strains capable of producing tetrodotoxin (TTX), it can only be achieved through chemical analysis of metabolites, which not only requires a large workload and a long cycle, but also the toxin standard used in chemical analysis is expensive and the analysis cost is high. , the present invention provides a method for rapid screening of microbial strains producing tetrodotoxin, which can quickly screen a large number of mixed bacterial strains through probe labeling

Method used

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  • A method for rapidly screening microbial strains producing tetrodotoxin and the used digoxin-labeled dna probe

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Embodiment 1

[0029] Sensitivity testing sample strains were cultivated, and samples were taken of Vibrio alginolyiicus VA (Vibrio alginolyiicus VA, whose metabolites were confirmed to produce TTX toxin by chemical analysis), and a single colony on the plate of the VA strain was picked, and its DNA sample was extracted according to the conventional method of molecular cloning, Dilute it 5-10 times.

Embodiment 2

[0031] For specific detection of sample strain cultivation, 2 strains of toxin-producing bacteria (A. Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Vibrio harveii, Vibrio salmonicida, Moraxella lacunae, P. The DNA samples were extracted according to the conventional method of molecular cloning, and diluted 5-10 times.

Embodiment 3

[0033]A method for rapidly screening microbial strains producing tetrodotoxin, 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 2) ...

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Abstract

The invention relates to the technical field of marine biology, in particular to a method for rapidly screening microbial strains producing tetrodotoxin and a digoxin-labeled DNA probe used therefor, the probe used in the method is 254 complementary to the DNA base to be tested Base-length single-stranded DNA, the 3' end of the probe is labeled with digoxigenin. Through colony in situ dot molecular hybridization, the probe can specifically detect whether there is tetrodotoxin synthesis sxt gene nucleic acid in the DNA sample of the strain to be tested, and screen out toxin-producing positive strains. The method involved in the invention utilizes the in situ extract of bacterial strain DNA to perform colony dot hybridization, and can complete the screening of 96 samples within 8-10 hours. The method of the invention is fast, has strong specificity and high accuracy.

Description

technical field [0001] The invention relates to the technical field of marine biology, in particular to a method for rapidly screening microbial strains producing tetrodotoxin and a digoxin-labeled DNA probe used therein. Background technique [0002] Tetrodotoxin (TTX) is a small molecule non-protein alkaloid neurotoxin that exists in pufferfish and other organisms. It is one of the most toxic neurotoxins found in nature. Its toxicity is more than a thousand times higher than that of cyanide, and it can block sodium ion channels on nerve excitatory membranes with high selectivity and high affinity, hinder nerve conduction, and cause nerve paralysis and death. TTX has a certain effect on headache, arthritis, tetanus, cholera, typhoid, asthma, whooping cough, advanced cancer and other diseases. Tetrodotoxin is expected to be developed as the preferred anesthetic and detoxification agent. As an analgesic, its analgesic effect is 3,000 times that of morphine, and 100,000 tim...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6841C12Q1/6834C12Q1/04C12N15/11
CPCC12Q1/6834C12Q1/6841C12Q2543/10C12Q2563/131
Inventor 杨桥张晓玲穆军蒋志伟张若男
Owner ZHEJIANG OCEAN UNIV
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