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A method for rapid screening of microbial strains producing gonitoxin and the used digoxin-labeled dna probe

The invention relates to a technology for labeling alginate toxin and digoxin, which can be used in biochemical equipment and methods, microbial determination/inspection, recombinant DNA technology, etc., and can solve the problems of rapid strain screening, high analysis cost, and large workload. Achieve the effect of not easy operation error, high efficiency and high precision

Active Publication Date: 2022-03-25
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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  • A method for rapid screening of microbial strains producing gonitoxin and the used digoxin-labeled dna probe

Examples

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Embodiment 1

[0029] Sensitivity detection sample strains were cultivated, and a sample of nitrate-reducing bacteria (Nitratereductor sp. LZ3-1, whose metabolites were chemically analyzed to confirm the production of GTXs toxin) was picked, and the single colony on the plate of the LZ3-1 strain was picked and extracted according to the conventional method of molecular cloning. DNA samples, diluted 5-10 times.

Embodiment 2

[0031]Specific detection of sample strain cultivation, take 1 toxin-producing bacteria (nitrate-reducing bacteria), 9 non-toxin-producing bacteria (including Escherichia coli, Shigella sohnsonii, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa) Monomonas, Vibrio harveii, Vibrio salmonicida, Moraxella lacullaria, Phomonas shigella), pick a single colony on the plate of each strain, and extract its DNA samples according to the conventional molecular cloning method, Dilute it 5-10 times.

Embodiment 3

[0033] A kind of method for quick screening to produce genus toxin microbial strain, described method comprises the following steps:

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

[0035] 2) Put the digoxigenin-labeled DNA into boiling water for 8 minutes and then cool it in an ice bath to obtain a labeled DNA solution. Add the labeled DNA solution to the pre-hybridization sol...

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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 gonitoxins and a digoxin-labeled DNA probe used therein. The probe used in the method is complementary to the DNA base to be tested 248 bases long 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 sxtS 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 genus toxin and a digoxigenin-labeled DNA probe used therefor. Background technique [0002] Gonyautoxins (GTXs), one of the paralytic shellfish toxins, are guanamine neurotoxins and are specific sodium channel blockers. GTXs have important applications in harmful red tide detection, neurophysiology, medical diagnosis, drug development, and biochemical warfare agents. In medical diagnosis and drug development, because of its unique chemical structure and toxicological mechanism of action, it has become a research cell Na + An important tool medicine for the channel. And it has a variety of effects such as high-efficiency analgesia, anesthesia, antispasmodic, antihypertensive, antiasthmatic, etc. It is an important leading compound for the development of new marine drugs. Some marine dinoflagellates, freshwater c...

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 张晓玲杨桥穆军蒋志伟张若男
Owner ZHEJIANG OCEAN UNIV
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