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Marine animal and human pathogenic bacterial-wound vibrion gene diagnostic reagent kit and detecting method

A technology for marine aquatic products and Vibrio vulnificus, which is applied in biochemical equipment and methods, determination/inspection of microorganisms, resistance to vector-borne diseases, etc., can solve the problems of troublesome preparation, limited application and development, and weak specificity, Achieve the effect of ensuring rapidity, avoiding the spread of germs, and high practical value

Inactive Publication Date: 2005-01-05
SUN YAT SEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some required materials are cumbersome to prepare, and the cost is high. Some methods are not sensitive and specific. Therefore, the techniques mastered by the majority of aquaculture owners and doctors are very difficult and difficult to promote, which limits the application and development of these techniques.

Method used

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  • Marine animal and human pathogenic bacterial-wound vibrion gene diagnostic reagent kit and detecting method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1: the genetic diagnosis kit of Vibrio vulnificus

[0031] The kit consists of the following parts (10 samples):

[0032] 1). Sample diluent (solution A), 1 tube, 5ml / tube, filled with 1×PBS, pH7.4.

[0033] 2).PCR reaction solution (solution B), 1 tube, 250μl / tube, containing PCR amplification reaction solution (25μl system), including ddH 2 O, containing Mg 2+ 10×Buffer, dNTP, primer VvF, primer VvR and TaqE.

[0034] 3). Positive control solution (solution C), 1 tube, 20 μl / tube, containing total DNA of Vibrio vulnificus, as a positive template;

[0035] 4). Cuboid box, 8.5×5.8×6.2cm 3 .

[0036] 5). A piece of foam board, the same size as the bottom of the box, 2.2cm high, four rows of holes, four holes in the first row, hole diameter 1.3cm, five holes in the second row, hole diameter 1.0cm, third and fourth There are six holes in each row, and the hole diameter is 0.6cm. The above-mentioned small tubes are respectively placed correspondingly in th...

Embodiment 2

[0047] Embodiment 2: detection method of marine aquatic animals and human pathogen-Vibrio vulnificus

[0048] Use the test kit of embodiment 1, carry out according to the following steps:

[0049]1). Using aseptic method, take 0.05 g of fresh abalone liver tissue, add 500 μl of sample diluent (liquid A) to dilute 10 times, and homogenize in an ice bath in a sterile homogenizer;

[0050] 2). Centrifuge at 6000r / min for 5min;

[0051] 3). Take 100 μl of the supernatant, boil for 15 minutes, and immediately put it on ice for 5 minutes;

[0052] 4). Centrifuge at 6000r / min for 10min, and use the supernatant as a PCR template;

[0053] 5). Take 1 μl of the template and solution C respectively, add it to the PCR reaction solution (solution B), mix well, centrifuge at 1000r / min for 10sec, and place it on the PCR instrument;

[0054] 6). Amplify according to the following conditions:

[0055] Pre-denaturation at 95°C for 3min→34 cycles at 94°C for 1min→10min at 72°C→storage at 4°C...

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Abstract

The invention provides a gene diagnosing reagent box and the detecting method of sea marine lives (such as shrimp, fish and so on) and human pathogenic bacterium-vibrio alginolyticus. There agent box and the detecting method are designed using a pair of primer as main body of vibrio alginolyticus gene protected area series design. The invention uses poly enzyme chain reaction (PCR) technology to carry on qualitative detection to the special DNA piece of the sea marine product and human pathogenic bacterium-vibrio, the method is quick, and the singularity is excellent, the sensitivity is high; it can be applied to the bacterium tracing and detecting to the sea marine product in each period, it also can be applied to the clinic detection of human intestinal canal acute contagion, and the environment detection and so on.

Description

technical field [0001] The invention relates to a diagnostic kit and a detection method for marine aquatic economic animals and human pathogenic bacteria, mainly for the genetic diagnosis of Vibrio vulnificus (Vibrio vulnificus). Background technique [0002] Vibrio vulnificus (Vibrio vulnificus) is a halophilic bacterium 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 and human food poisoning caused by Vibrio vulnificus in coastal areas of my country. It is currently known that Vibrio vulnificus can infect many types of aquatic products such as barramundi, European eel, Japanese eel, tilapia, Chinese prawn, eel, grouper, oyster and wrinkled abalone in Australia; Con...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/33
CPCY02A50/30
Inventor 邓先余何建国王智学黄志坚翁少萍
Owner SUN YAT SEN UNIV
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