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Reagent kit for rapid detection of vibrio harveyi and detection method thereof

A technology of vibrio harveyi and kits, which is applied in the field of kits for rapid detection of vibrio harvelii, and can solve the problems of low sensitivity, high cost, and difficulty in popularization

Inactive Publication Date: 2008-06-18
GUANGDONG OCEAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The preparation of some required materials is troublesome and expensive, and some methods are not sensitive and specific. Therefore, the techniques mastered by medical staff and aquaculture owners are very difficult and difficult to promote, which limits the application and development of these techniques.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] A kit for rapid detection of Vibrio harveyi was made according to the following formula:

[0031] Reagent A: 0.1M NaCl, 10mM Tris.Cl (pH8.0), 1mM EDTA (pH8.0), 5% TritonX-100;

[0032] Reagent B: 10mg / ml lysozyme, 10mM Tris.Cl (pH8.0);

[0033] Reagent C: 5.0 μl 10 x PCR Buffer, 0.5 μl TaqE (concentration 5 U / μl), 35.5 μl ddH 2 O;

[0034] Reagent D: 4.0 μl of NTP (2.5 mM each), 3.0 μl of MgCl 2 (25mM), 10pM VhF, 10pM VhR;

[0035] Reagent E: 50x TAE;

[0036] Reagent F: agarose (1%) + ethidium bromide (0.5 μg / ml).

[0037] Follow the procedure below:

[0038]1. Sample processing and DNA extraction: take about 0.1-0.5 g of shrimp tissue, add 400 μl of reagent A to the sample, mash it with a sterilized toothpick, centrifuge at 12,000 g for 2 minutes, discard the supernatant, and re-use 350 μl of reagent A and 50 μl of reagent B. Suspend the precipitate and mix well. Let stand at room temperature for 5 minutes, take a water bath at 100°C for 5 minutes, and cool to...

Embodiment 2

[0044] A kit for rapid detection of Vibrio harveyi was made according to the following formula:

[0045] Reagent A: 0.3M NaCl, 10mM Tris.Cl (pH8.0), 1mMEDTA (pH8.0), 5% TritonX-100;

[0046] Reagent B: 5mg / ml lysozyme, 10mM Tris.Cl (pH8.0);

[0047] Reagent C: 2.5 μl 10 x PCR Buffer, 0.25 μl TaqE (concentration 5 U / μl), 35.5 μl ddH 2 o

[0048] Reagent D: 2.0μldNTP (2.5mM each), 1.5μlMgCl 2 (25mM), 10pM VhF, 10pM VhR;

[0049] Reagent E: 50x TAE;

[0050] Reagent F: agarose (1.2%) + ethidium bromide (0.8 μg / ml).

[0051] Follow the procedure below:

[0052] 1. Sample processing and DNA extraction: Take about 0.1-0.5 grams of fish tissue, add 200 μl of reagent A to the sample, mash it with a sterilized toothpick, centrifuge at 12,000 g for 2 minutes, discard the supernatant, and re-use 150 μl of reagent A and 50 μl of reagent B. Suspend the precipitate and mix well. Let stand at room temperature for 5 minutes, take a water bath at 100°C for 5 minutes, and cool to room t...

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PUM

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Abstract

The invention provides a kit and a detection method for rapidly detecting harveyi Vibrioharveyi. The kit and the detection method are designed, taking a pair of primers which are designed in harveyi Vibrioharveyi heat-resistant direct gyrB gene conservative area sequence as a main body. The invention adopts the polymerase chain reaction (PCR) technology and implements the qualitative detection on the specific DNA fragment of marine aquatic pathogen, the harveyi Vibrioharveyi. The invention has the advantages of convenient and rapid properties, good specificity and high sensitivity and can be applied to both the harveyi Vibrioharveyi tracking detection during the culture processes of various periods of aquatic animals and the environmental monitoring. With high practical value, the invention can avoid the transmission and the prevalence of germs.

Description

technical field [0001] The invention relates to a kit for rapidly detecting Vibrio harveyi, and further relates to a method for rapidly detecting Vibrio harveyi using the kit for rapidly detecting Vibrio harveyi. Background technique [0002] Vibrio harveyi (V.harveyi) is a luminescent vibrio that is widely distributed in coastal waterways, bays and coastline areas and seafood all over the world. This bacterium is an opportunistic pathogenic bacterium, which can break out and become popular under certain conditions. In recent years, with the deterioration of the breeding environment, there have been more and more reports of seafood disease or mass death caused by Vibrio harveyi in coastal areas of my country. At present, it is known that Vibrio harveii can infect many types of aquatic products such as sea bass, large yellow croaker, giant tiger prawn, meji prawn, grouper, etc., which brings great harm to the aquaculture industry. Bacterial diseases caused by Vibrio harveli...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04G01N27/447G01N21/33
Inventor 孙成波周银环
Owner GUANGDONG OCEAN UNIVERSITY
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