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

A Digoxigenin labeling and DNA probe technology, applied in the field of marine biology, can solve the problems of heavy workload, high analysis cost, and long cycle, and achieve the effect of low error, high efficiency, and not easy to operate error

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

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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] Specific detection sample strain cultivation, take samples of 2 strains of toxin-producing bacteria (Vibrio alginolyticus, Alteromonas tetrodotoxin), 9 strains of non-toxin-producing bacteria (including Escherichia coli, Shigella sonneri, Golden Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Vibrio harveii, Vibrio salmonicida, Moraxella lacunae, P. The DNA samples were extracted according to the conventional methods 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 organisms and in particular relates to a method for rapidly screening a microorganism strain capable of generating tetrodotoxin 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 254 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 sxt gene nucleic acid or not, and a toxin-producing positive strain is screened. According to the method provided 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 inventionis rapid, high in specificity and high in 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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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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