Method for screening paralytic shellfish poisoning generation strain by using DNA probe-bacterial colony in-situ hybridization technique

A DNA probe and in situ hybridization technology, applied in biochemical equipment and methods, microbiological determination/inspection, etc., can solve the problems of expensive toxin standard products, high analysis cost, heavy workload, etc., and achieve high market prospects With economic value, simple operation steps and high accuracy

Active Publication Date: 2018-02-27
ZHEJIANG OCEAN UNIV
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AI Technical Summary

Problems solved by technology

However, the workload is heavy, the cycle is long, and the toxin standards used in chemical analysis are expensive and

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] The DNA probe-colony in situ hybridization technique screens the method for producing bacterial strains of paralytic shellfish poisoning, and the sensitivity test of this method comprises the following steps:

[0023] 1) Select the bacterium Herfleria ( Hoeflea sp.) (CCTCC AB 2016294) is the sample to be tested, and the chemical analysis of its metabolites confirms the production of PSP toxin;

[0024] 2) Pick a single colony on the plate of the Heffleria strain, extract its DNA sample according to the conventional method of molecular cloning, and dilute it by 2-10 times;

[0025] 3) Take 2 μL of the diluted sample and spot it on a nitrocellulose filter, soak it in 0.5M NaOH solution for 10 minutes, dry it naturally, sandwich it between two pieces of dry filter paper, place it in a vacuum oven, bake at 80°C for 2 hours, and fix the DNA; Put lysozyme in TE buffer at an amount of 5 mg / mL, put it into the DNA membrane for 30 minutes at 37°C, and then rinse with TE buffe...

Embodiment 2

[0030] DNA probe-colony in situ hybridization method for screening paralytic shellfish virus-producing strains, the samples used are: 2 strains of toxin-producing bacteria: including Sulfite and Heffleria; 9 strains of non-toxin-producing bacteria: including Escherichia coli , Shigella sonneri, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Vibrio harveii, Vibrio salmonicida, Moraxella lacunae, Pseudomonas shigella-like. The specificity test of this method comprises the following steps:

[0031] Take a single colony of the strain to be tested on a nitrocellulose filter, soak it in 0.5M NaOH solution for 10 minutes, dry it naturally, sandwich it between two pieces of dry filter paper, place it in a vacuum oven, bake it at 80°C for 2 hours, and fix it. DNA: Add lysozyme to TE buffer according to the amount of 5 mg / mL, put it into the DNA membrane for 30 minutes at 37°C, rinse with TE buffer to remove bacterial cell residues.

[0032] Place the DNA membrane pr...

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PUM

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Abstract

The invention discloses a method for screening paralytic shellfish poisoning generation strains by using a DNA probe-bacterial colony in-situ hybridization technique. The method comprises steps of pretreatment, hybridization and screening. By using specific forward primers and reverse primers, a single chain DNA probe marked by digoxin is synthesized through polymerase chain reaction amplification, DNA of a strain to be tested is hybridized with the DNA probe, and poisoning generation strains are screened according to developing testing hybridization matching results. The method has the beneficial effects that 90 samples can be screened within 8-10 hours by using the method disclosed by the invention, the method is rapid and efficient, good in specificity and high in accuracy, the method is simple in operation step, small in workload and relatively short in cycle, reagents and materials used in the method are all common reagents for biochemical tests, and thus the method is harmless tohuman bodies, environmental-friendly, low in analysis cost, relatively good in market prospect and high in economic value.

Description

technical field [0001] The invention belongs to the technical field of marine organisms, and in particular relates to a DNA probe-colony in situ hybridization technique for screening paralytic shellfish virus-producing bacterial strains. technical background [0002] Among the known red tide toxins, paralytic shellfish posoning (PSP) is the most toxic, widely distributed, most threatening, and causes the most frequent poisoning events. It is mainly composed of Saxitoxin (STX) and its derivatives. It belongs to guanamine toxoids and is a typical sodium ion channel blocker. There are as many as 58 PSP homologues that have been discovered so far. Its biosynthetic genes are sxt gene clusters. STX has important applications in red tide detection, molecular biology, neurobiology, medical diagnosis, drug development, military biochemical weapons and other research. In terms of medical diagnosis and drug development, the unique chemical structure and toxicological mechanism of S...

Claims

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Application Information

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IPC IPC(8): C12Q1/6841C12Q1/689
CPCC12Q1/6841C12Q1/689C12Q2600/158
Inventor 张晓玲杨桥穆军
Owner ZHEJIANG OCEAN UNIV
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