Reagent case for diagnosing gene of pathogenic bacterial and para hematolysis vibrion of marine water product animal and hunman and testing method thereof

A technology for marine aquatic products and Vibrio hemolyticus, applied in biochemical equipment and methods, measurement/testing of microorganisms, and resistance to vector-borne diseases, etc., can solve problems such as limited application and development, troublesome preparation, and low sensitivity, and avoid Spread of germs, high practical value, and rapid effect

Inactive Publication Date: 2005-01-05
SUN YAT SEN UNIV +1
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AI Technical Summary

Benefits of technology

This patented technology allows for quicker and more accurate identification of fish or other seafood organisms that may have been exposed during their development stages through testing them against specific microorganism strains found naturally occurring within our environment (the gut flora). By comparing this data from different samples taken at similar times we aimed towards finding out what types were causing these harmful agents. Overall, it provides technical benefits over current techniques such as culture analysis and molecular diagnostic tests.

Problems solved by technology

There were many examples where fish had contracted from saltwater into fresh water due to various factors like salinity changes during their migration across seawater regions such as Antarcoda Island and Mediterranean Sea. However, current treatments against harmful organisms including V. Parasuisa cannot effectively protect them because they may develop resistance towards drugs used to control other species' growth. Current tests involve complicated procedures requiring highly specialized equipment, making it hard to apply at home without significant risk of contamination.

Method used

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  • Reagent case for diagnosing gene of pathogenic bacterial and para hematolysis vibrion of marine water product animal and hunman and testing method thereof

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Embodiment 1

[0031] Embodiment 1: the genetic diagnosis kit of Vibrio parahaemolyticus

[0032] The kit consists of the following parts (10 samples):

[0033] 1). Sample diluent (solution A), 1 tube, 5ml / tube, filled with 1×PBS, pH7.4.

[0034] 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, primers VpF, VpR and TaqE.

[0035] 3).Positive control solution (solution C), 1 tube, 20μl / tube, containing the total DNA of Vibrio parahaemolyticus, as a positive template;

[0036] 4). Cuboid box, 8.5×5.8×6.2cm 3 .

[0037] 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 correspondin...

Embodiment 2

[0048] Embodiment 2: Detection method of marine aquatic animals and human pathogenic bacteria-Vibrio parahaemolyticus

[0049] Use the test kit of embodiment 1, carry out according to the following steps:

[0050] 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;

[0051] 2). Centrifuge at 6000r / min for 5min

[0052] 3). Take 100 μl of the supernatant, boil for 15 minutes, and immediately put it on ice for 5 minutes;

[0053] 4). Centrifuge at 6000r / min for 10min, and use the supernatant as a PCR template;

[0054] 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 machine;

[0055] 6). Amplify according to the following conditions:

[0056] Pre-denaturation at 95°C for 3min→34 cycles at 94°C for 1min→10min at 72°C→st...

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Abstract

The invention provides a gene diagnosis reagent box and its detecting method for sea aquatic animal (like Chinese turtle, tilapia, eel, Hyriopsis cumingii, etc) and human pathogens-Vibrio parahaemolyticus, designed by using a pair of primers designed by the sequence in conservative region of Vibrio parahaemolyticus gene to act as main body. It adopts the polyase chain reaction (PCR) technique to qualitatively detect the specific DNA fragments of the Vibrio parahaemolyticus, simply and conveniently, and rapidly, good-specificity, and high-sensitivity; and can be used in bacteria tracking detection of aquatic animal pathogens in the course of breeding in each period, and also the clinic detection of human intestinal acute infections, environmental monitoring, avoiding the pathogens infecting and popularizing and it has very great practical value.

Description

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Claims

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Application Information

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Owner SUN YAT SEN UNIV
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