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 accurate method and convenient use

Inactive Publication Date: 2014-07-09
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Summary
  • 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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Embodiment 1

[0030] 1) Determination of degenerate primers:

[0031] The protein sequences of various known bacterial catalases were collected from GenBank, and after multiple sequence alignments, the conserved amino acid sequences were found to obtain suitable degenerate primers for PCR amplification of the catalase gene. Four kinds, two forward primers and two reverse primers,

[0032] The forward primer sequence (5' to 3') is:

[0033] C3+:TTTYAAYCGAGARMGRGTNCCNGA;

[0034] CA:GTACCTGAAMGRGTRGTSCAYGCNMRRGG;

[0035] The reverse primer sequence (5' to 3') is:

[0036] CB: GAATATGCAAAAAGGCGNCCYTGNARNAKYTTRTC;

[0037] C3-: AATCTTTATGAGGCCAAACTTTTGTNANRTCRAA.

[0038]2) Use the forward degenerate primer CA:GTACCTGAAMGRGTRGTSCAYGCNMRRGG and the reverse degenerate primer C3-:AATCTTTATGAGGCCAAACTTTTGTNANRTCRAA to amplify the catalase gene fragment by PCR. Use 12.5 microliters of reaction system for PCR amplification: 1 microliter of 2.5mM dNTP, 1.25 microliters of 10×Taq PCR amplificati...

Embodiment 2

[0043] 1) Using forward degenerate primer CA: GTACCTGAAMGRGTRGTSCAYGCNMRRGG and reverse degenerate primer CB: GAATATGCAAAAAGGCGNCCYTGNARNAKYTTRTC, PCR amplifies the catalase gene fragment. Use 12.5 microliters of reaction system for PCR: 1 microliter of 2.5mM dNTP, 1.25 microliters of 10×Taq PCR buffer, 0.15 microliters of each of the two degenerate primers at 50 μM, 0.625 U of Taq DNA polymerase, 1 microliter of seawater DNA, and add water to Total volume 12.5 microliters. Six 12.5 microliter PCR systems were used each time to prepare each catalase gene fragment. PCR conditions: 94°C for 2min; 94°C for 30s, 60°C→51°C for 1min, 72°C for 1min30s, 10 cycles, each cycle reducing the annealing temperature by 1°C; 94°C for 30s, 50°C for 1min, 72°C for 1min, 20 cycles; 72°C 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 c...

Embodiment 3

[0048] 1) Use forward degenerate primer C3+: TTTYAAYCGAGARMGRGTNCCNGA and reverse degenerate primer CB: GAATATGCAAAAAGGCGNCCYTGNARNAKYTTRTC to amplify the catalase gene fragment. Use 12.5 microliters of reaction system for PCR: 1 microliter of 2.5mM dNTP, 1.25 microliters of 10×Taq PCR buffer, 0.15 microliters of each of the two degenerate primers at 50 μM, 0.625 U of Taq DNA polymerase, 1 microliter of seawater DNA, and add water to Total volume 12.5 microliters. Six 12.5 microliter PCR systems were used each time to prepare each catalase gene fragment. PCR conditions: 94°C for 2min; 94°C for 30s, 60°C→51°C for 1min, 72°C for 1min30s, 10 cycles, each cycle reducing the annealing temperature by 1°C; 94°C for 30s, 50°C for 1min, 72°C for 1min, 20 cycles; 72°C 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.

[0049] 2) Use the T vector kit to connect the...

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Abstract

The invention relates to a method for detecting the diversity of bacterial catalase in seawater by using degenerate primers, designing a combination of four kinds of degenerate primers based on the conserved region of bacterial catalase, and using polymerase chain reaction to amplify the diversity of bacterial catalase in seawater. Catalase gene fragments, and then use Escherichia coli to construct a library of enzyme gene fragments, use restriction endonucleases to digest the enzyme genes in the library, detect the molecular weight distribution of the fragments by electrophoresis, and determine the types of different electrophoresis bands, and finally Different types of catalase genes were sequenced separately to obtain information on the diversity of catalase genes. This method is an accurate, reliable, rapid, new method using degenerate primers to detect the diversity of bacterial catalase in seawater, and can be used to detect the diversity of bacterial catalase in seawater.

Description

technical field [0001] The invention belongs to the field of marine microbial ecology, in particular 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, which is highly oxidizing and harmful. Catalase can rapidly decompose hydrogen peroxide and protect biomolecules from oxidative damage; catalase is an important housekeeping protein that keeps active in the life activities of aerobic microorganisms. [0003] Low-temperature environments exist widely 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 the influence of reactive oxygen species. Catalase is an antioxidant enzyme necessary for the metabolism of aerobic cryogenic organi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
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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