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Kit for fast test of vibrio harveyi and the test method thereof

A technology of vibrio harveyi and a kit, which is applied in the field of kits for rapid detection of vibrio harveyi, which can solve the problems of time-consuming, unreliable detection results, and unreliable results, etc.

Inactive Publication Date: 2004-09-08
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

(References: "Practical Clinical Bacteriology Examination and Progress" edited by Lou Yongxin Wang Jinliang, 1992 Tianjin Science and Technology Translation Publishing Company), the results of this method are more accurate but very time-consuming, and the detection results of new species are not very reliable; 2, API-20E and other rapid bacterial identification kits, which use statistical analysis to draw identification conclusions after detecting a few biochemical indicators, relatively save time, but the results are not reliable

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] A kit for rapid detection of Vibrio harveyi was made according to the following formula:

[0028] Reagent A: 0.1M NaCl, 10mM Tris.Cl (pH8.0), 1mM EDTA (pH8.0), 5% Triton X-100

[0029] Reagent B: 10mg / ml lysozyme, 10mM Tris.Cl (pH8.0)

[0030] Reagent C: 5.0μl 10×PCR Buffer, 0.5μl Taq (5U / μl), 35.5μl ddH 2 o

[0031] Reagent D: 4.0μl dNTP (2.5mM each), 3.0μl MgCl 2 (25mM), 20pM VP 01 20pM VP 02

[0032] Reagent E: 50×TAE

[0033] Reagent F: agarose (1%) + ethidium bromide (0.5 μg / ml)

[0034] Follow the procedure below for testing:

[0035]1. Sample processing and DNA extraction: Take about 0.1-0.5 grams of fish 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. Place at room temperature for 5 minutes, then in a water bath at 100°C for 5 minutes. Cool to room temperature, ...

Embodiment 2

[0041] Various solutions were prepared according to the following recipes:

[0042] Reagent A: 0.3M NaCl, 10mM Tris.Cl (pH8.0), 1mM EDTA (pH8.0),

[0043] Reagent B: 5mg / ml lysozyme, 10mM Tris.Cl (pH8.0)

[0044] Reagent C: 2.5μl 10×PCR Buffer, 0.25μl Taq (5U / ul), 35.5μl ddH 2 o

[0045] Reagent D: 2.0μl dNTP (2.5mM each), 1.5μl MgCl 2 (25mM), 10pM VP 01 (0.5μl), 10pM VP 02 (0.5μl)

[0046] Reagent E::50×TAE

[0047] Reagent F: agarose (1.2%) + ethidium bromide (0.8 μg / ml)

[0048] Follow the procedure below:

[0049] 1. Sample Processing and DNA Extraction

[0050] Take about 0.1-0.5 g 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, resuspend the pellet with 150 μl of reagent A and 50 μl of reagent B, and mix well. Place at room temperature for 5 minutes, then in a water bath at 100°C for 1 minute. Cool to room temperature and centrifuge at 12000g for 10 minut...

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Abstract

The present invention is kit for fast test of vibrio Harveyi and the test method thereof. The kit includes DNA extracting reagent comprising buffering reagent A and cell cracking reagent B; PCR reagent comprising reaction pre-mixing reagent C and reaction primer reagent D with specific primer pair of VP01 5'-GCACCATGGGTTAAAAGCCGCT and VP02 5'-AGAGAACTGACGCACAACGTGGTTC; and electrophoresis and development reagent comprising reagent E and reagent F. The test method includes: sample processing, DNA extraction, PCR proliferation, electrophoresis and development test. Under ultraviolet light test, one tested 480 nucleotide pair stripe corresponding to the infection or pollution of vibrio Harveyi, or else no infection or pollution of vibrio Harveyi. The present invention has the advantages of fast detection and high sensitivity.

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. technical background [0002] In recent years, my country's mariculture industry has developed rapidly, and the output of mariculture has increased by 5-6 times in the past 10 years. However, the large-scale development of mariculture in the previous stage was very blind and even destructive, which led to the slowdown in the growth of mariculture production in the past two years, especially the problem of disease has become more and more serious. Among them, vibriosis is one of the important pathogens that harm a variety of mariculture organisms such as fish, shrimp, shellfish and algae. Vibrio is an indigenous bacterium of marine origin, widely distributed in seawater and marine organisms; pathogenic Vibrio can cause explosive death of cultured organisms, and can ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/00C12Q1/68G01N33/50G01N33/561G01N33/569G01N33/68
Inventor 陈偿胡超群
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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