PCR rapid detection method for marine culturable bacteria, and kit

A technology of marine bacteria and kits, applied in the field of microbial detection, to achieve the effects of increasing yield, shortening extraction time, and increasing specificity

Inactive Publication Date: 2019-01-18
HEZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

And there is no specific kit for rapid PCR of marine bacteria

Method used

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  • PCR rapid detection method for marine culturable bacteria, and kit
  • PCR rapid detection method for marine culturable bacteria, and kit

Examples

Experimental program
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Effect test

Embodiment 1

[0045] PCR rapid test of cultivable bacteria in South China Sea sediments

[0046] S1. Sample processing

[0047] Put 10g of the marine sediment sample taken from the South China Sea into 80ml sterile seawater with glass beads, incubate at 28℃, 140rpm shaker for 30min, then transfer 1ml suspension into 9ml sterile seawater and mix well. 10 -1 -10 -5 Five different concentration gradients. Using the spreading plate method, 0.1ml samples with different concentration gradients were spread on the entire solid medium, four in parallel, two by one placed in an incubator at 28°C and 10°C for 2-7 days. Purification is performed according to the morphological characteristics of the colony after culture.

[0048] extract

[0049] Pipette 100 µL of 10% (w / v) sterile Chelex-100 solution into a 1.5mL Eppendorf tube, use a sterilized toothpick to pick out the purely cultured colonies, pick the size of the rice grains to the tube wall, crush and disperse Dissolve and mix the Chelex-100 solution. ...

Embodiment 2

[0058] Rapid PCR Test of Symbiotic Actinomyces from Gorgonian Coral in the North Sea

[0059] S1. Sample processing

[0060] Gorgonians to be collected in the North Sea Anthogorgia caerulea Take 10g of the sample and grind it with a mortar and put it into a 100ml triangular bottle with sterile seawater, take it out after 2 days in a shaker at 28°C and 140 rpm, and take the supernatant to prepare 10 -1 -10 -5 At different dilutions, draw 0.5 ml of each multiple of dilutions and spread on the solid culture, and incubate at 28°C for 3-5 days. After being cultured, the colonies are first distinguished by naked eyes according to the characteristics of the colonies, marked, and then further observed with a microscope to determine the separation object, and then a single colony is picked with a sterilized bamboo stick, and the quartering method is repeated for separation and purification.

[0061] DNA extraction of symbiotic actinomycetes

[0062] After the purified strain, a certain amount...

Embodiment 3

[0071] A kit for rapid PCR of marine cultivable bacteria, which is characterized by comprising: marine bacterial DNA extraction reagents, PCR reaction reagents, and electrophoresis reagents, wherein:

[0072] (1) Marine bacterial DNA extraction sample preparation and extraction reagent concentration: colonies of marine cultivable bacteria purely cultured in seawater medium, 10% (w / v) sterile Chelex-100 solution;

[0073] (2) Each raw material and its concentration in the PCR reaction reagent: 16S rDNA universal primer (27F, 1492R), 10×PCRBuffe, 5U / Taq enzyme, 10Mm dNTP and ddH 2 O; Among them, the universal primer sequences are 27F and 1492R in the prior art, and their sequences are:

[0074] 27F: 5ʹ-AGAGTTTGATCC TGGCTCAG-3ʹ;

[0075] 1492R: 5ʹ-GGTTACCTTGTTACGA CTT-3ʹ.

[0076] (3) Electrophoresis reagents: agarose, 50×TBE, Goldview II nucleic acid dye, DNA Marker;

[0077] A method for rapid PCR with a kit for rapid PCR detection of marine cultivable bacteria is characterized in that it...

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Abstract

The invention discloses a PCR rapid detection method for marine culturable bacteria, and a kit, belonging to the technical field of microorganism detection. The kit comprises a DNA extraction reagent,a PCR reaction reagent and an electrophoretic reagent. Among them, DNA extraction reagent is Chelex- 100; The PCR reaction reagents included two universal 16S rRNA primers (27F and 1492R) and 2 x TaqPCR Mastermix solution. Electrophoretic reagents included agarose, 50 x TBE, Goldview II nucleic acid dye, DNA Marker. The kit can be used for DNA amplification of marine culturable bacteria in a short time, and has the advantages of simple procedure, safety, economy, high efficiency and small bacterial requirement.

Description

Technical field [0001] The invention relates to the technical field of microbial detection, in particular to a method and kit for rapid PCR detection of marine cultivable bacteria. Background technique [0002] As the research on terrestrial resources tends to be normalized and the available materials are becoming less and less, the existing microbial molecular ecology research shows that there are a large number of microbial resources in the ocean, which are very important for the earth's material cycle, ecological balance and human health. Significance, therefore, more domestic and foreign researchers have focused on the ocean. Because the unique environment of the ocean has endowed marine bacteria with novel, purposeful and diversified metabolically active substances, there are more and more researches on marine bacteria. The identification of bacteria is an important content of microbial research and a necessary research method to analyze the diversity of its unique physiolo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686
CPCC12Q1/686C12Q1/689C12Q2565/125
Inventor 杨小梅谷毅鹏段振华伍淑婕陈伟玲
Owner HEZHOU UNIV
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