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Rapid screening method of microorganisms capable of producing sea anemone toxins

A screening method and microbial technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of high analysis cost, heavy workload, expensive toxin standard products, etc., and achieve strong operability

Active Publication Date: 2021-01-05
舟山出入境检验检疫局综合技术服务中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The key technology is the rapid screening of toxin-producing strains, which can be achieved mainly through chemical analysis of metabolites. This method not only has a large workload and a long cycle, but also the toxin standard used in chemical analysis is expensive and the analysis cost is high.
At present, there is no rapid screening technology for anemone toxin-producing strains, so it is of great significance to explore effective rapid screening methods for anemone toxin-producing microorganisms

Method used

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  • Rapid screening method of microorganisms capable of producing sea anemone toxins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1 (sensitivity test of the present invention)

[0043] (1) Sample: Marine bacteria Nioella sp.LZ7-4 (CCTCC AB 2017231) was purchased from China Center for Type Culture Collection, and chemical analysis of its metabolites confirmed toxin production.

[0044] (2) Sample processing: Pick a single colony on the plate of the LZ7-4 strain, extract its genomic DNA sample according to the conventional method of molecular cloning, and dilute it 8 times for later use.

[0045] (3) Obtaining bacterial strain DNA: take 1 μL and 2 μL of the above bacterial DNA samples and spot them on 6*6cm 2 The nitrocellulose filter membrane was soaked in 0.42M NaOH solution for 6 minutes, and the membrane was taken out and baked in an oven at 60°C for 1 hour to fix the bacterial DNA. Then place it in a solution of lysozyme (5 mg / mL, pH 8.2) and incubate at 37°C for 15 min. Bacterial cell residues on the membrane surface were washed away with TE buffer.

[0046] (4) Obtain DNA probe ...

Embodiment 2

[0050] Embodiment 2 (sensitivity test of the present invention)

[0051] (1) Sample: Marine bacteria Nioella sp.LZ7-4 (CCTCC AB 2017231) was purchased from China Center for Type Culture Collection, and chemical analysis of its metabolites confirmed toxin production.

[0052] (2) Sample processing: Pick a single colony on the plate of the LZ7-4 strain, extract its genomic DNA sample according to the conventional method of molecular cloning, and dilute it 5 times for later use.

[0053] (3) Obtaining bacterial strain DNA: take 1 μL and 2 μL of the above bacterial DNA samples and spot them on 6*6cm 2 The nitrocellulose filter membrane was immersed in 0.4M NaOH solution for 7 minutes, and the membrane was taken out and baked in an oven at 62°C for 1.1 hours to fix the bacterial DNA. Then place it in a solution of lysozyme (5.1 mg / mL, pH 8.3) and incubate at 36°C for 14 minutes. Bacterial cell residues on the membrane surface were washed away with TE buffer.

[0054] (4) Obtain ...

Embodiment 3

[0058] Embodiment 3 (sensitivity test of the present invention)

[0059] (1) Sample: Marine bacteria Nioella sp.LZ7-4 (CCTCC AB 2017231) was purchased from China Center for Type Culture Collection, and chemical analysis of its metabolites confirmed toxin production.

[0060] (2) Sample processing: Pick a single colony on the plate of the LZ7-4 strain, extract its genomic DNA sample according to the conventional method of molecular cloning, and dilute it 10 times for later use.

[0061] (3) Obtaining bacterial strain DNA: take 1 μL and 2 μL of the above bacterial DNA samples and spot them on 6*6cm 2 Dip the nitrocellulose filter membrane in 0.44M NaOH solution for 8 minutes, take out the membrane and bake it in an oven at 65°C for 1.2 hours to fix bacterial DNA. Then placed in lysozyme (5.2mg / mL, pH 8.4) solution, incubated at 38°C for 18min. Bacterial cell residues on the membrane surface were washed away with TE buffer.

[0062] (4) Obtain DNA probe hybridization solution:...

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Abstract

The invention relates to the technical field of marine organisms, aims at the problem of the technological gap of a rapid screening method of microorganisms capable of producing sea anemone toxins, and discloses a rapid screening method of microorganisms capable of producing sea anemone toxins. The rapid screening method comprises the following steps: (1) acquiring bacterial strain DNA; (2) obtaining DNA probe hybridization solution, i.e., after placing digoxin labeled DNA probe solution into boiling water, placing the digoxin labeled DNA probe solution into an ice bath, and taking the DNA probe solution to add into hybridization solution; (3) carrying out hybridization, i.e., placing a DNA membrane into the DNA probe hybridization solution, carrying out incubation and hybridization, and placing the hybridized DNA membrane into a container containing a 2*SSC solution for washing; (4) developing; (5) carrying out result determination. The DNA probe and bacterial colony in-situ hybridization method is used for rapid screening of microbial strains capable of producing the sea anemone toxins; the strain capable of producing the sea anemone toxins are accurately screened out through theDNA probes subjected to specific amplification and simple and ordered operation steps; and the method is simple, efficient, short in screening period and high in accuracy.

Description

technical field [0001] The invention relates to the technical field of marine organisms, in particular to a rapid screening method for sea anemone toxin-producing microorganisms. Background technique [0002] Palytoxins (PLTXs) are one of the most toxic non-protein algal toxins known, and are listed as one of the largest non-polymeric natural products; they are highly toxic, fat-soluble, heat-stable polyether marine biotoxins ; Is one of the most widely distributed marine toxins with the highest incidence. It can cause irreversible damage to various organs and tissues such as intestines, liver, nerves, and placenta, and there is no specific medicine to treat it after poisoning. Sea anemone toxins seriously endanger the safety of marine ecosystems and human health, and have become a worldwide research hotspot. Sea anemone toxin is a neurotoxin, which has important applications in the detection of harmful red tides, neurophysiology, medical diagnosis, drug development, and bi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6841C12Q1/04
CPCC12Q1/6841C12Q2527/125C12Q2563/131C12Q2565/518
Inventor 张静周秀锦邵宏宏胡兴娟
Owner 舟山出入境检验检疫局综合技术服务中心
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