Method for rapid identification of Edwardsiella fisicida sRNA

A fast, suicide plasmid technology, applied in the direction of biochemical equipment and methods, microbial determination/inspection, etc., to achieve the effect of easy wide application, high-throughput detection and identification, and reduced detection cost

Active Publication Date: 2019-01-25
INST OF TROPICAL BIOSCI & BIOTECH CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the research on sRNA mainly focuses on the pathogenic bacteria of humans, mamm

Method used

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  • Method for rapid identification of Edwardsiella fisicida sRNA
  • Method for rapid identification of Edwardsiella fisicida sRNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0035] Embodiment 2 real-time fluorescent quantitative PCR analysis

[0036] In this example, real-time fluorescent quantitative PCR was used to analyze the expression difference of sRNA082 between the wild strain and the mutant strain.

[0037] Methods: The wild strain of Edwardsiella piscicida and the hfq mutant strain were cultured to the logarithmic phase (OD=0.8), the bacteria were collected, and the RNA was extracted with the EZNA Total RNA Extraction Kit from Omega Company, and the RNA was treated with RNase-free DNaseI. Take 1 μg of RNA sample, and use Invitrogen’s Superscript II reverse transcriptase to perform reverse synthesis of cDNA; use cDNA as a template for real-time fluorescent quantitative PCR: the reagent is Takara PrimeScript TM RT-PCR KitII; reaction parameters: pre-denaturation at 95°C for 30s, denaturation at 95°C for 10s, annealing and extension at 60°C for 30s, 40 cycles; sRNA primers (taking Edwardsiella ichthyophila sRNA012 as an example), 082RTF:AA...

Embodiment 3

[0039] Embodiment 3 Accuracy detection test

[0040] Using the method of Example 1 of the present invention and the real-time fluorescent quantitative PCR experiment conditions of Example 2, 19 sRNA samples were identified, among which, 19 samples were determined to be Hfq-dependent sRNA by transcriptome sequencing. see results figure 2 .

[0041] The results show that the method provided by the present invention detects the above 19 samples, and determines that 16 samples are Hfq-dependent sRNA, and three samples of sR063, sR009, and sR162 are Hfq-dependent sRNA, with an accuracy rate of 84.2%. It shows that the method provided by the invention can quickly and accurately identify the dependence relationship between sRNA and Hfq protein.

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Abstract

The invention relates to the field of molecular biotechnology, in particular to a rapid identification method of Edwardsiella fisicida sRNA. The method provided by the invention comprises the following steps: constructing an Edwardsiella fisicida hfq gene deletion mutant strain; the expression levels of sRNA in wild strain and hfq mutant were detected by fluorescence quantitative PCR, and the dependency of sRNA on Hfq protein was determined according to the difference of expression levels between wild strain and hfq mutant. The method provided by the invention can quickly and accurately determine the dependency relationship between the sRNA and the Hfq protein, has simple operation steps, does not need the high technical difficulty experiment operation such as RNA immunoprecipitation and the like, can realize the high-throughput detection and identification, greatly reduces the detection cost, and is easy to be widely applied.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method for rapidly identifying sRNA of Edwardsiella piscicida. Background technique [0002] Edwardsiella piscicida, formerly known as Edwardsiella tarda, is an important pathogenic bacteria in aquatic products, which can infect flounder, turbot, eel, red sea bream, salmon, tilapia Diseases such as fish hemorrhagic septicemia and necrosis of visceral tissues are collectively referred to as Edwardsiellosis (edwardsiellosis). The disease is prevalent and harmful all over the world, and has caused huge economic losses to the aquaculture industry. Although the research on the pathogenicity of Edwardsiella piscicida has made positive progress, a variety of pathogenicity-related factors and systems have been discovered and studied, and its genome sequence map has also been completed, the pathogenesis of Edwardsiella piscicidae is still unclear. It is clear how it adapts t...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686
CPCC12Q1/686C12Q1/689C12Q2563/107C12Q2545/114
Inventor 胡永华黄惠琴杜和禾
Owner INST OF TROPICAL BIOSCI & BIOTECH CHINESE ACADEMY OF TROPICAL AGRI SCI
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