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Kit and detection method for rapidly detecting multiple virulence factors GeXP of vibrio harveyi

A detection kit and technology for Vibrio harveyi, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of inability to fully reflect the potential pathogenicity of strains, single detection content, etc., to improve accuracy , detect the effect of wide coverage

Inactive Publication Date: 2012-11-21
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in terms of pathogenicity detection of Vibrio harveyi, the defects of these methods are that the detection content is too single and cannot fully reflect the potential pathogenicity of the strain

Method used

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  • Kit and detection method for rapidly detecting multiple virulence factors GeXP of vibrio harveyi
  • Kit and detection method for rapidly detecting multiple virulence factors GeXP of vibrio harveyi
  • Kit and detection method for rapidly detecting multiple virulence factors GeXP of vibrio harveyi

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Experimental program
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Effect test

Embodiment 1

[0055] (1) Extraction of Vibrio harveyi genomic DNA

[0056] Firstly, the Vibrio harveyi in the sample was purified and isolated.

[0057] ① Add 50 μL of 10% (w / v) sterile Chelex-100 solution into the centrifuge tube;

[0058] ②Pick a single colony from the culture plate of Vibrio harveyi into the above Chelex-100 solution;

[0059] ③ Place the centrifuge tube on a vortex mixer and shake it fully for 10 seconds, take a boiling water bath for 10 minutes, cool to room temperature, and shake it fully for another 10 seconds;

[0060] ④ Centrifuge at 13000r / min for 2min, and the supernatant is the genomic DNA of Vibrio harveyi, and store it in a -20°C refrigerator for later use.

[0061] (2) Design and synthesis of multigene PCR primers

[0062] Primers designed for the virulence gene hlyA, the amplified fragment size is about 147bp:

[0063] hlyA-F: 5'-AGGTGACACTATAGAATATAGATGATGACAGCACGGGA-3'

[0064] hlyA-B: 5'-GTACGACTCACTATAGGGAGGTTGACCACTCACGGAAAT-3';

[0065] Primers d...

Embodiment 2

[0137] (1) Preparation of Vibrio harveyi genomic DNA

[0138] Firstly, the Vibrio harveyi in the sample was purified and isolated.

[0139] ①Add 90 μL 10% (w / v) sterile Chelex-100 solution into the centrifuge tube;

[0140] ②Pick a single colony from the culture plate of Vibrio harveyi into the above Chelex-100 solution;

[0141] ③ Place the centrifuge tube on a vortex mixer and shake it fully for 15 seconds, take a boiling water bath for 8 minutes, and then shake it fully for 10 seconds after cooling to room temperature;

[0142] ④ Centrifuge at 8000r / min for 20min, and the supernatant is the genomic DNA of Vibrio harveyi, and store it in a -20°C refrigerator for later use.

[0143] (2) Design and synthesis of primers

[0144] Primers designed for the virulence gene hlyA, the amplified fragment size is about 147bp:

[0145] hlyA-F: 5'-AGGTGACACTATAGAATATAGATGATGACAGCACGGGA-3'

[0146] hlyA-B: 5'-GTACGACTCACTATAGGGAGGTTGACCACTCACGGAAAT-3';

[0147] Primers designed for t...

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Abstract

The invention discloses a kit and a detection method for rapidly detecting multiple virulence factors GeXP of vibrio harveyi. A genome DNA (deoxyribonucleic acid) of the vibrio harveyi (V. Harveyi) is firstly extracted to serve as a template, twelve pairs of polygene PCR (polymerase chain reaction) primers and a pair of universal primers are used for multiple PCR amplification to obtain multiple PCR reaction products, and the products are detected and analyzed by a Genome Lab GeXP genetic analysis system. Specific primers are designed according to virulence factors of twelve known types of vibrio harveyi, twelve genes are rapidly and efficiently detected once by means of single-tube reaction to know whether detected samples contain the vibrio harveyi carrying the virulence factors and having potential pathogenicity or not, detection coverage is wide, accuracy of evaluating pathogenicity of the vibrio harveyi can be greatly improved, more effective detection measures are provided for evaluating pathogenicity of the vibrio harveyi, and the detection method has important application prospects in aquaculture disease detection, epidemiological investigation, detection of pathogenic bacteria in food, aquatic product import and export detection, hospital infection monitoring and the like.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and in particular relates to a GeXP rapid detection kit and detection kit for multiple virulence factors used for detecting pathogenic Vibrio harveyi (Vibrio harveyi, VH.) in clinical examination, public health and food inspection. method. Background technique: [0002] Vibrio harveyi (V.Harveyi), also known as Vibrio harveyi, is a Gram-negative bacterium that is facultatively anaerobic, respiratory and fermentative, and has luminescent properties. The bacterium is widely distributed in the marine environment and marine organisms, and can cause disease in seawater fish and other farmed animals. It is a pathogenic bacterium of shrimp and fish cultured in many countries and regions in the world. One of the main pathogenic bacteria, the pathogen has caused huge economic losses to the mariculture industry. [0003] Once vibriosis breaks out, it is difficult to control. The conventional detection metho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 陈偿邓益琴高磊任春华胡超群
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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