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Gene chips for detecting multiple pathogenic bacteria in animals cultivated in sea water and uses thereof

A gene chip and mariculture technology, which is applied to the gene chip and application field for detecting various pathogenic bacteria of marine aquaculture animals, can solve the problems of lack of evidence and persuasiveness in the virulence analysis of pathogenic bacteria and so on.

Inactive Publication Date: 2014-08-13
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

However, the existing chips mainly use highly conserved ribosomal sequences among genera or sequences of certain characteristic genes as probes, and are committed to the accurate identification of pathogenic bacteria, but there is no favorable evidence and persuasiveness in the virulence analysis of pathogenic bacteria.

Method used

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  • Gene chips for detecting multiple pathogenic bacteria in animals cultivated in sea water and uses thereof
  • Gene chips for detecting multiple pathogenic bacteria in animals cultivated in sea water and uses thereof
  • Gene chips for detecting multiple pathogenic bacteria in animals cultivated in sea water and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0087] Then follow the following specific steps for chip preparation, sample preparation, chip hybridization, and acquisition and analysis of hybridization results:

[0088] 1. Chip preparation:

[0089] Dissolve the probe modified with the 3'end amino group to a final concentration of 10-100μM, mix it with 30-60% DMSO (dimethyl sulfoxide), and dilute it into a spot sample with a final probe concentration of 10-30μM. The spotter performs the spot system of the chip, and the layout of the probe array on the chip is shown in Table 3. After the probe is spotted and fixed according to the instructions of the aldehyde-based or amino-based substrate fixing process.

[0090] Second, the application of the chip

Embodiment 1

[0091] Example 1: Effect detection of the chip

[0092] (1) Extraction of DNA from samples to be tested:

[0093] Randomly select 8 species of pathogenic bacteria in marine aquaculture animals: Vibrio anguillarum, Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio fischeri, Vibrio splendidu, Aeromonas hydrophila, Aeromonas salmonicida, Edwards tardy Bacteria, and calf thymus DNA are tested for the effect of gene chip.

[0094] Use liquid culture medium to cultivate pathogenic bacteria overnight at an appropriate temperature, use bacterial genomic DNA extraction kit or traditional methods to extract bacterial genomic DNA, and dilute it to 1.0ng / μL as a PCR reaction template.

[0095] Randomly select 14 characteristic genes of 8 pathogens and 3 genes of the quality control system for synchronous gene chip testing. The primers and probes corresponding to these 17 genes are as follows:

[0096] Table 5. Primers and probes information corresponding to 14 genes of 8 mariculture animal pat...

Embodiment 2

[0120] Example 2: Application of the chip

[0121] (1) Extraction of DNA from samples to be tested

[0122] The gill tissue of a diseased turbot of a farm in Shandong was taken, half of which was extracted using the marine animal tissue genomic DNA extraction kit produced by Tiangen Biochemical Technology Co., Ltd.; the other half was used for bacterial analysis on 2216E seawater medium Cultivation and single colony screening, retain one of the dominant bacteria, and use the bacterial genomic DNA extraction kit to extract the genomic DNA of the bacteria, which is also used for gene chip detection.

[0123] (2) PCR synchronous amplification of target sequences of characteristic genes and quality control system genes:

[0124] Use all the primers in Table 5 and the 16S rRNA primers in Table 6 to simultaneously amplify the template to be tested, and add a separate primer pair to each PCR tube. The amplification reaction solution includes genomic DNA from the gill tissue of turbot, 1.0μL...

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Abstract

The invention provides a gene chip for detecting various seawater cultured animal pathogenic bacteria and its application. The pathogenic bacteria include: Vibrio anguillarum, Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio fischeri, Vibrio splendidus, Vibrio riverina, Vibrio vulnificus, Vibrio cholerae non-O1, Vibrio mimicus, Vibrio salmonicida, Aeromonas hydrophila, Aeromonas sobria, Aeromonas salmonicida, Edward tarda Bacillus, Edwardsiella catfish, Renalobacter salmonidum, Mycobacterium marinum, Pseudomonas, Streptococcus.

Description

Technical field [0001] The invention relates to a detection technology of pathogenic bacteria in mariculture animals, in particular to a gene chip for detecting pathogenic bacteria of various mariculture animals and its application. Background technique [0002] Bacterial diseases have an important impact on the health of mariculture animals and the quality and safety of aquatic products. There are many types of bacteria that can cause diseases in mariculture animals. Simultaneous infection of multiple microorganisms often occurs. The traditional bacterial detection methods are mostly physiological and biochemical identification and 16S. The combination of rRNA sequencing, but the operation is cumbersome and takes a long time, and it is impossible to judge the pathogenicity of microorganisms. Subsequent detection methods developed include indirect fluorescent antibody technology, nucleic acid blot hybridization, PCR detection methods, etc., but none of them can meet the demand fo...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/689
Inventor 黄倢李晨杨冰王秀华荆晓艳张庆利张宝存赵培谢国驷闫丽
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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