Combined metabolic marker and detection kit for judging genetic modification effect of pathogenic bacteria of visceral white-spot disease of epinephelus coioides

A technology for visceral white spot disease and oblique grouper, which is applied in biological testing, measuring devices, material inspection products, etc., and can solve the problems of cumbersome and time-consuming, and limited methods for judging the effect of genetic modification.

Pending Publication Date: 2022-02-01
JIMEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the complexity of the pathogenic mechanism, the methods for judging the effect of genetic modification are relatively limited. Considering the tedious and time-consuming pathological examination, an accurate and stable method is developed for judging the pathogenicity of visceral white spot disease in the oblique-banded grouper. The genetic modification effect of bacteria is of great significance
At present, there is no report on the combination of the three lipid metabolites to judge the intervention effect of the gene clpV of grouper visceral white spot disease

Method used

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  • Combined metabolic marker and detection kit for judging genetic modification effect of pathogenic bacteria of visceral white-spot disease of epinephelus coioides
  • Combined metabolic marker and detection kit for judging genetic modification effect of pathogenic bacteria of visceral white-spot disease of epinephelus coioides
  • Combined metabolic marker and detection kit for judging genetic modification effect of pathogenic bacteria of visceral white-spot disease of epinephelus coioides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Spleen sample collection: Spleen samples of grouper in WT group and clpV-RNAi group were collected at different time points (6, 12, 24, 48, 72, 96 hpi) after infection. At each time point, 7 fish were randomly sacrificed in each group, and about 15 mg of spleen samples were weighed.

[0038] Analytical method

[0039] Spleen sample pretreatment: put 15 mg of fresh spleen tissue into a 2 ml centrifuge tube, add 300 μL methanol extractant containing internal standard, and use a ball mill (mixer mill MM400, Restch Technology, Han, Germany) to homogenize the tissue (20 Hz, 3 × 1 min ). Then add 1000 μL MTBE and vortex at room temperature for 20 min, then add 300 μL ultrapure water and vortex at room temperature for 30 s, let stand at 4 °C for 5 min, and then centrifuge at high speed (10,000 rpm, 8 °C, 10 min) to separate the two phases and quantitatively collect the upper hydrophobic phase And vacuum-dried in a refrigerated centrifugal concentrator, and the obtained lyoph...

Embodiment 2

[0051] Spleen sample collection: collect grouper spleen samples from WT group and PBS group, and the sampling method is the same as in Example 1.

[0052] Analytical method

[0053] The methods of spleen sample pretreatment and mass spectrometry analysis are the same as those in Example 1.

[0054] Result analysis: the purpose of this example is to verify whether the change trends of the above three markers in the PBS control group are comparable to those in the clpV-RNAi group, so as to further confirm the effect of clpV gene silencing.

[0055] Compared with the WT group, the content of phosphatidylethanolamine (18:2) and hydroxylated fatty acid (38:2) in the PBS group was significantly down-regulated, and the content of phosphatidylinositol (39:4) was significantly increased, and the trend of change was similar to that of clpV-RNAi Group Concordance ( figure 2 & image 3 ). The WT and PBS group samples at all time points were included in the discriminant comparison, an...

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Abstract

The invention discloses a combined metabolic marker and a detection kit for judging the genetic modification effect of pathogenic bacteria of visceral white-spot disease of epinephelus coioides, and relates to lipid metabolites such as phosphatidyl ethanolamine (18:2), hydroxylated fatty acid (38:2) and phosphatidylinositol (39:4) in spleen samples of epinephelus coioides as combined markers, and the method is used for judging the effect of silencing of the pseudomonas proteiniflora virulence gene clpV causing the grouper visceral white-spot disease on the grouper. The invention also relates to a kit for judging the gene modification effect. The method comprises the following steps: detecting the relative concentration of lipid metabolites in a spleen sample of grouper infected by pseudomonas proteinus, calculating a combined marker variable P based on a binary logistic regression equation, and determining a cut point value, and judging the condition of the pathogenic bacteria of the visceral white-spot disease of the grouper after genetic modification. The kit has the characteristics of high sensitivity, high detection efficiency, low cost and good repeatability, and has a good application prospect.

Description

technical field [0001] The invention relates to the fields of analytical chemistry, biochemistry and fishery science, in particular to a combined metabolic marker and a detection kit for judging the genetic modification of the pathogenic bacteria of white spot disease in the viscera of grouper. Background technique [0002] Pseudomonas plecoglossicida, also known as Pseudomonas plecoglossicida, is a widely distributed aerobic Gram-negative bacillus. The fungus can cause visceral white spot disease of large yellow croaker (Larimichthyscrocea), slanted grouper (Epihepheluscoioides) and other economic marine fish, leading to a large number of farmed fish deaths in a short period of time, resulting in serious economic losses. The interaction between the host and pathogenic bacteria is complex, and the infection process involves a large number of dynamic changes such as gene expression and metabolic interference. These interactions are critical for understanding the host's abili...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06G01N30/72G01N33/92
CPCG01N30/02G01N30/06G01N30/72G01N33/92
Inventor 曾珺钟月鄢庆枇
Owner JIMEI UNIV
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