Vibrio shilonii multiple virulence factor GeXP rapid detection kit and detection method thereof

A technology of Vibrio schlossere and detection kit is applied in the field of rapid detection kits for pathogenic Vibrio-Vibrio schlosserii, can solve the problems of cumbersome process, time-consuming detection, low detection sensitivity, etc., and achieves wide detection coverage, The effect of improving accuracy

Active Publication Date: 2014-08-27
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods have certain defects: (1) the detection is time-consuming and the process is cumbersome; (2) the detection sensitivity is low, and the pathogenic bacteria are often detected in the late stage of the disease; (3) the target gene for detection is too single, which is easy to miss due to the environment Different strains of the same species caused by differences cannot fully reflect the potential pathogenicity of the pathogenic bacteria

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  • Vibrio shilonii multiple virulence factor GeXP rapid detection kit and detection method thereof
  • Vibrio shilonii multiple virulence factor GeXP rapid detection kit and detection method thereof
  • Vibrio shilonii multiple virulence factor GeXP rapid detection kit and detection method thereof

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Embodiment 1

[0056] (1) Genomic DNA extracted from Vibrio shilonii

[0057] Firstly, the Vibrio shilonii in the coral sample was purified and isolated on a plate, and then the Vibrio shilonii was cultured.

[0058] ① Add 50 μL of 10% (w / v) sterile Chelex-100 solution to the centrifuge tube;

[0059] ②Pick a single colony from the culture plate of Vibrio shilonii into the above Chelex-100 solution;

[0060] 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;

[0061] ④ Centrifuge at 12000r / min for 2min, and the supernatant is the genomic DNA of Vibrio shilonii, and store it in a -20°C refrigerator for later use.

[0062] (2) Design and synthesis of primers for multigene PCR

[0063] Primers designed for the virulence gene sod (NCBI Reference Sequence: NZ_ABCH01000140.1), the amplified fragment size is about 110bp:

[0064] sod-F: AGGTGACACTATAGAATA ...

Embodiment 2

[0141] (1) Genomic DNA extracted from Vibrio shilonii

[0142] Firstly, the Vibrio shilonii in the coral sample was purified and isolated on a plate, and then the Vibrio shilonii was cultured.

[0143] ① Add 50 μL 10% (w / v) sterile Chelex-100 solution to the centrifuge tube;

[0144] ②Pick a single colony from the culture plate of Vibrio shilonii into the above Chelex-100 solution;

[0145] 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;

[0146] ④ Centrifuge at 12000r / min for 2min, and the supernatant is the genomic DNA of Vibrio shilonii, and store it in a -20°C refrigerator for later use.

[0147] (2) Design and synthesis of primers for multigene PCR

[0148] Primers designed for the virulence gene sod (NCBI Reference Sequence: NZ_ABCH01000140.1), the amplified fragment size is about 110bp:

[0149] sod-F: AGGTGACACTATAGAATA CCG...

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Abstract

The present invention discloses a Vibrio shilonii multiple virulence factor GeXP rapid detection kit and a detection method thereof. According to the present invention, genome DNA of Vibrio shilonii is extracted and is adopted as a template, 13 pairs of multi-gene PCR primers and a pair of universal primers are adopted to carry out multiplex PCR amplification to obtain multiplex PCR reaction products, and a GenomeLab GeXP genetic analysis system is adopted to carry out detection analysis. With the present invention, the special primers are designed based on the 13 Vibrio shilonii virulence genes, a single-tube reaction is performed to rapidly and efficiently detect the 13 genes in one time so as to obtain the following results, the detection coverage is wide, and the Vibrio shilonii detection accuracy can be substantially improved, wherein the results comprise whether the detected sample contains the virulence carrying gene and the Vibrio shilonii having the potentially pathogenic ability. In addition, a certain theory basis is provided for coral bleaching phenomenon prevention and control, and important practical significances are provided for further coral island protection in our country and further degradation prevention.

Description

Technical field: [0001] The invention relates to the field of biotechnology, in particular to a rapid detection kit and detection method specially used for Vibrio shilonii, a pathogenic Vibrio that can cause coral bleaching. Background technique: [0002] Vibrio shilonii is a marine vibrio that can infect corals and cause bacterial bleaching of corals. At present, the detection methods of Vibrio schrouhei are mainly traditional microbial classification and identification methods and detection methods relying on PCR technology. Traditional microbial classification and identification methods are mainly based on the morphological, physiological and biochemical characteristics of microorganisms. Although the results are highly reliable, they are cumbersome and time-consuming. At this stage, the most widely used method is molecular biology, which mainly relies on PCR technology to amplify certain specific genes. However, these methods have certain defects: (1) the detection is ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCY02A50/30
Inventor 陈偿高磊任春华胡超群
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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