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Gene chip for detecting pathogenic vibrio flora in sea cucumber, shrimp and shellfish culture regions and use method

A pathogenic Vibrio and gene chip technology is applied in the field of gene chips for detecting pathogenic Vibrio groups in ginseng, shrimp and shellfish breeding areas, and can solve the problems of false positives, affecting the accuracy of test results, and easy to produce deviations.

Inactive Publication Date: 2017-06-09
DALIAN OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the probes designed for existing gene chips are aimed at the 16S rRNA gene, and 16S rDNA is relatively conservative for the detection of pathogenic Vibrio. Due to the lack of information sites, the stability of the constructed system is poor, and it is used to identify genetic relationships. Closer bacteria are prone to bias, causing false positives and affecting the accuracy of test results

Method used

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  • Gene chip for detecting pathogenic vibrio flora in sea cucumber, shrimp and shellfish culture regions and use method
  • Gene chip for detecting pathogenic vibrio flora in sea cucumber, shrimp and shellfish culture regions and use method
  • Gene chip for detecting pathogenic vibrio flora in sea cucumber, shrimp and shellfish culture regions and use method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Design and synthesis of embodiment 1 probe

[0032] 1. Target detection strains and target genes

[0033] The present invention selects important common pathogenic Vibrio groups in the environment and organisms of ginseng and shellfish breeding areas as detection objects, including Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio anguillarum, Vibrio harveyi, Vibrio vulnificus, Vibrio brilliant. Vibrio parahaemolyticus uses thermostable direct hemolysin and multidrug resistance protein as target genes, Vibrio alginolyticus uses 23rDNA as target gene, Vibrio anguillarum uses methyl-accepting chemotaxis protein as target gene, Vibrio harveii uses glycolipid transfer protein as target gene For genes, Vibrio vulnificus uses methyl-accepting chemotaxis protein as the target gene, and Vibrio resplendent uses putative permeases of the drug / metabolite transporter as the target gene.

[0034] 2. Probe design

[0035] The present invention uses 18-30bp oligonucleotide prob...

Embodiment 2

[0044] The making of embodiment 2 gene chip

[0045] Utilize the probe designed in Example 1 to prepare a gene chip for detecting important common pathogenic Vibrio groups in the environment and organisms of the ginseng shellfish culture area, and the specific steps are as follows:

[0046] 1. Preparation of probe mother solution

[0047] Dilute the synthesized probe (Table 2) into a 100 μmol / L mother solution with double distilled water, and then dilute it to a concentration of 20 μmol / L with a spotting buffer;

[0048] 2. Preparation before sampling

[0049] Transfer 100 μL to wells A1, B1, C1, D1, E1, F1, G1, H1, I1, and J1 of a 96 / 384-well plate, and add 100 μL of spotting buffer to well K1;

[0050] 3. Spotting

[0051] Use the AD1500 gene chip spotting instrument of Bio-Dot Company of the United States to carry out non-contact spotting on the aldehyde substrate according to the pre-set program; the spotting volume of each point is 0.5 μL, the spot diameter is 200 μm, ...

Embodiment 3

[0059] For the method of using the important common pathogenic Vibrio gene chip (prepared in embodiment 2) in the environment and organisms of the ginseng shellfish breeding area, the specific steps are as follows:

[0060] 1. Collection and processing of samples to be tested

[0061] Use an 8 μm sterile filter membrane to remove impurities from the obtained water sample, then filter it with a 0.22 μm filter membrane and collect the filter membrane, wrap it in sterile aluminum foil, and store it at -20°C;

[0062] The mud sample is taken from 100g of sediment at the bottom 2-5cm of the breeding environment, and the collected samples are transported to the laboratory in an ice box;

[0063] Animal samples were cut with sterile scissors, rinsed with sterile water three times, put into cryopreservation tubes, and stored in liquid nitrogen.

[0064] 2. Template DNA preparation

[0065] DNA from water samples and mud samples was extracted with OMEGA Water DNA Kit (OMEGA Water DNA...

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Abstract

The invention discloses a gene chip for detecting pathogenic vibrio flora in sea cucumber, shrimp and shellfish culture regions, and a use method. The gene chip comprises a chip carrier, wherein chemically modified nucleotide probes for detecting vibrio parahaemolyticus, vibrio alginolyticus, vibrio anguillarum, vibrio harveyi, vibrio vulnificus and vibrio splendidus are immobilized on the chip carrier. The use method comprises the following steps: DNA extraction of a sample to be detected, PCR amplification of the sample, hybridization of a PCR product with a chip, processing after hybridization of the chip, scanning of hybridization chip, and interpretation of results. The gene chip significantly improves the detection specificity by specific probes designed for each vibrio variation region sequence, improves the capability of identifying pathogenic vibrio flora of environments and organisms of the sea cucumber, shrimp and shellfish culture regions, and also greatly shortens the detection time.

Description

technical field [0001] The invention belongs to the field of microorganism detection, and in particular relates to a gene chip for detecting pathogenic Vibrio group in ginseng shellfish breeding areas and a method for using it. Background technique [0002] With the rapid development of artificial breeding and the ecological changes of the breeding waters, vibriosis has become one of the main bacterial diseases of marine animals. Vibriosis (Vibriosis), caused by Vibrio bacteria, is the most prevalent and most harmful bacterial disease in aquatic animals of the ginseng shellfish cultured around the world, causing serious economic losses to the aquaculture industry. Studies have shown that Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio anguillarum, Vibrio harveyi, Vibrio vulnificus and Vibrio splendidus in the Vibrio genus are dominant in the Vibrio population and widely exist in natural seawater. It leads to the occurrence of vibriosis, and has the characteristics of ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C40B40/06
CPCC12Q1/6837C12Q1/689C12Q2600/16C40B40/06C12Q2537/143C12Q2531/113C12Q2565/501Y02A50/30
Inventor 丁君王荦常亚青
Owner DALIAN OCEAN UNIV
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