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Reagent kit for fast detection of vibrio proteolyticus and its detection process

A technology of vibrio proteolyticus and kits, which is applied in biochemical equipment and methods, measuring devices, microbe determination/inspection, etc. It can solve cumbersome and time-consuming problems, and achieve high sensitivity, convenient detection, and good specificity Effect

Inactive Publication Date: 2007-06-06
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the techniques for identification of Vibrio include: 1. Classical physiological and biochemical identification methods, which are cumbersome, time-consuming, and require operators to have good professionalism; 2. Bacteria rapid automatic identification systems, which require expensive instruments and equipment and Skilled operators; 3. Nucleic acid-based detection technology, including DNA probe and PCR technology (polymerase chain reaction technology), has the characteristics of fast, simple and sensitive compared with the above-mentioned detection technology, but has not yet See Patents for PCR Technology and Detection Kit for Detection of Vibrio Proteolyticus

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  • Reagent kit for fast detection of vibrio proteolyticus and its detection process
  • Reagent kit for fast detection of vibrio proteolyticus and its detection process

Examples

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Effect test

Embodiment 1

[0033] Example 1 A kit for rapid detection of Vibrio proteolyticus was made according to the following formula:

[0034] (a) Reagent A (enrichment medium): contains tryptone, beef extract, yeast extract and agar powder in a weight ratio of 5:3:1:18.

[0035] (b) Polymerase chain reaction (25 μl system) reagents: including reaction premix reagent B and reagent C,

[0036] Reagent B contains 10x PCR buffer (containing MgCl 2 15mM), specific primer pair VpS and VpA, dNTP (a mixture of four deoxynucleotides), bovine serum albumin and sterile double distilled water, the contents are 2.5μl, 0.5μl (10μM), 0.5μl (10μM) , 0.5 μl (10 μM each), 0.5 μl (5 mg / ml) and 19.375 μl.

[0037] Reagent C: Taq enzyme, 0.125 μl (5 units / μl).

[0038] (c) Electrophoresis and chromogenic reagents, including reagent D, reagent E and reagent F.

[0039] Reagent D: 50 times TAE (electrophoresis buffer), containing trihydroxymethylaminomethane-acetic acid and ethylenediaminetetraacetic acid, the conc...

Embodiment 2

[0047] Example 2 A kit for rapid detection of Vibrio proteolyticus was made according to the following formula:

[0048] (a) Reagent A (enrichment medium): containing tryptone, beef extract, yeast extract and agar powder in a weight ratio of 5:3:1:18.

[0049] (b) Reagents for polymerase chain reaction (50 μl system): including reaction premix reagent B and reagent C. Reagent B contains 10x PCR buffer (with MgCl 2 15mM), specific primer pair VpS and VpA, dNTP (a mixture of four deoxynucleotides), bovine serum albumin and sterile double distilled water, the contents are 5.0μl, 1.0μl (10μM), 1.0μl (10μM) respectively , 1.0 μl (10 μM each), 1.0 μl (5 mg / ml) and 38.75 μl.

[0050] Reagent C: Taq enzyme, 0.25 μl (5 units / μl).

[0051] (c) Electrophoresis and chromogenic reagents, including reagent D, reagent E and reagent F.

[0052] Reagent D: 50 times TAE (electrophoresis buffer), containing trihydroxymethylaminomethane-acetic acid and ethylenediaminetetraacetic acid, the co...

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Abstract

The present invention provides one kind of reagent kit for fast detection of Vibrio proteolyticus. The reagent kit includes enrichment culture medium, PCR reagent, and electrophoresis and color developing reagent. The present invention also provides the method of fast detecting Vibrio proteolyticus with the reagent kit, and the detecting method has no step of extracting bacteria DNA and lower detection limit as low as 10<2> bacteria, and is fast, accurate and sensitive. The primer of the present invention has excellent specificity and produces no amplification signal to detected sample without Vibrio proteolyticus. The present invention may be applied for detecting various kinds of sample, including raised animal and raising water.

Description

technical field [0001] The invention relates to a kit for rapidly detecting Vibrio proteolyticus and a method for rapidly detecting Vibrio proteolyticus using the kit. Background technique [0002] Vibrio is the main pathogen of marine animals, and it has been found that Vibrio proteolyticus is one of the pathogens that cause a large number of deaths in near river oysters. Monitoring Vibrio proteolyticus in farmed animals and seawater is an important measure to prevent and control the diseases caused by it. At present, the techniques for identification of Vibrio include: 1. Classical physiological and biochemical identification methods, which are cumbersome, time-consuming, and require operators to have good professionalism; 2. Bacteria rapid automatic identification systems, which require expensive instruments and equipment and Skilled operators; 3. Nucleic acid-based detection technology, including DNA probe and PCR technology (polymerase chain ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/04C12Q1/68G01N27/447G01N21/33
Inventor 张其中张占会
Owner JINAN UNIVERSITY