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Method utilizing degenerate primer to detect diversity of bacteria catalase in seawater

A technology of bacterial catalase and catalase, which is applied in the direction of biochemical equipment and methods, and the determination/inspection of microorganisms, can solve the problem of time-consuming, expensive, and undeveloped catalase diversity detection methods and other problems, to achieve the effect of convenient use and precise method

Inactive Publication Date: 2013-01-02
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the activity of catalase in seawater can be directly detected, but the type and content of catalase cannot be studied directly, so the diversity information cannot be obtained; constructing a metagenomic library and sequencing can obtain the peroxidase activity in the environment. Hydrogenase gene information, but it is expensive and time-consuming, so there is no relevant report; using specific conserved primers to amplify target genes in environmental sample DNA is an ideal method for studying environmental gene diversity
There are currently no conserved primers for amplifying catalase genes in the environment, and therefore no corresponding method for detecting catalase diversity has been developed

Method used

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  • Method utilizing degenerate primer to detect diversity of bacteria catalase in seawater

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Experimental program
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Effect test

Embodiment 1

[0030] 1) Determination of degenerate primers:

[0031] Collect various known bacterial catalase protein sequences from GenBank and perform multiple sequence comparisons to find out the conservative amino acid sequences, thereby obtaining suitable degenerate primers for PCR amplification of catalase genes. There are four kinds of forward primers and two reverse primers. The sequence of forward primers (5' to 3') is:

[0032] C3+: TTTYAAYCGAGARMGRGTNCCNGARMGNG;

[0033] CA: GTACCTGAAMGRGTRGTSCAYGCNMRRGG;

[0034] The reverse primer sequence (5’ to 3’) is:

[0035] CB: GAATATGCAAAAAGGCGNCCYTGNARNAKYTTRTC;

[0036] C3-:AATCTTTATGAGGCCAAACTTTTGTNANRTCRAA. ,

[0037] 2) Use forward degenerate primer CA:CCNGARMGNGTNGTNCAYGCN and reverse degenerate primer C3-:

[0038] AATCTTTATGAGGCCAAACTTTTGTNANRTCRAA, PCR amplification of catalase gene fragment. PCR amplification uses 12.5 μl reaction system: 2.5 mM dNTP 1 μl, 10×Taq PCR amplification buffer 1.25 μl, 50 μM two degenerate primers each 0.15 μ...

Embodiment 2

[0043] 1) Use forward degenerate primer CA:CCNGARMGNGTNGTNCAYGCN and reverse degenerate primer CB:AAATGTTCGGCCYTGNARNADYTTRTC to amplify catalase gene fragments by PCR. PCR uses 12.5 μl reaction system: 2.5 mM dNTP 1 μl, 10×Taq PCR buffer 1.25 μl, 50 μM two degenerate primers each 0.15 μl, Taq DNA polymerase 0.625U, seawater DNA 1 μl, Add water to a total volume of 12.5 microliters. Six 12.5 microliter PCR systems were used to prepare each catalase gene fragment. PCR conditions: 94℃2min; 94℃30s, 60℃→51℃1min, 72℃1min 30s, 10 cycles, each cycle reduces the annealing temperature by 1℃; 94℃30s, 50℃1min, 72℃1min, 20 cycles; 72 ℃ 3min. After the reaction, 6 tubes of PCR products were combined, and a single PCR product with a molecular weight of 500bp to 1000bp was purified using a gel recovery kit.

[0044] 2) Use the T vector kit to connect the PCR products of step 1) and transform E. coli competent cells to construct a T vector library of catalase gene fragments. Blue-white scree...

Embodiment 3

[0048] 1) Use forward degenerate primer C3+:TTTYAAYCGAGARMGRGTNCCNGA and reverse degenerate primer CB:AAATGTTCGGCCYTGNARNADYTTRTC to amplify catalase gene fragments. PCR uses 12.5 μl reaction system: 2.5 mM dNTP 1 μl, 10×Taq PCR buffer 1.25 μl, 50 μM two degenerate primers each 0.15 μl, Taq DNA polymerase 0.625U, seawater DNA 1 μl, Add water to a total volume of 12.5 microliters. Six 12.5 microliter PCR systems were used to prepare each catalase gene fragment. PCR conditions: 94℃2min; 94℃30s, 60℃→51℃1min, 72℃1min 30s, 10 cycles, each cycle reduces the annealing temperature by 1℃; 94℃30s, 50℃1min, 72℃1min, 20 cycles; 72 ℃ 3min. After the reaction, 6 tubes of PCR products were combined, and a single PCR product with a molecular weight of 500 bp to 1000 bp was purified using a gel recovery kit.

[0049] 2) Use the T vector kit to connect the PCR products of step 1) and transform E. coli competent cells to construct a T vector library of catalase gene fragments. Blue-white screen...

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Abstract

The invention relates to a method utilizing degenerate primer to detect the diversity of bacteria catalase in seawater. Four types of combination of degenerate primer are designed in a bacteria catalase conserved region; catalase gene segments in seawater total DNA are amplified through polymerase chain reaction; then a library of the catalase gene segments is established through coliform bacteria; the catalase gene segments in the library are subjected to enzyme digestion through restriction enzyme; the molecular weight distribution of the segments is detected through cataphoresis, so as to determine different types of electrophoretic bands; and finally different types of the catalase genes are respectively subjected to order-checking, so that information of the diversity of catalase gene can be obtained. The method is accurate, reliable and rapid, uses degenerate primer to detect the diversity of bacteria catalase in seawater, and can be used for detecting the diversity of bacteria catalase in seawater.

Description

Technical field [0001] The invention belongs to the field of marine microbial ecology, and particularly relates to a method for detecting the diversity of bacterial catalase in seawater by using degenerate primers. Background technique [0002] Hydrogen peroxide is produced during life activities, especially during aerobic metabolism. Hydrogen peroxide is a kind of active oxygen with strong oxidizing property and great harm. Catalase can quickly decompose hydrogen peroxide and protect biomolecules from oxidative damage; catalase is an important housekeeping protein that remains active in the life of aerobic microorganisms. [0003] Low-temperature environments are widespread in the ocean. Under low-temperature conditions, the solubility of oxygen increases, and the increase in oxygen concentration makes low-temperature microorganisms more susceptible to active oxygen. Catalase is an antioxidant enzyme necessary for the metabolism of aerobic hypothermic organisms. The marine cryo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/30
Inventor 王伟孙谧纪晓峰袁翠
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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