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Kit for diagnosing gene of pathogenic bacterial and river vibrion of aquatic animal and human and testing method thereof

A fluvibrio vibrio and aquatic animal technology, applied in the field of gene diagnosis kits and detection of fluvibrio vibrio, can solve the problems of limited application and development, troublesome preparation, weak specificity, etc., so as to avoid the spread of germs and ensure fast speed The effect of 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 technology mastered by the majority of aquaculture owners is very difficult and difficult to promote, which limits the application and development of these technologies.

Method used

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  • Kit for diagnosing gene of pathogenic bacterial and river vibrion of aquatic animal and human and testing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[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 VfF, primer VfR and TaqE.

[0034] 3). Positive control solution (solution C), 1 tube, 20 μl / tube, containing the total DNA of Vibrio fluvialus, 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 t...

Embodiment 2

[0047] Embodiment 2: detection method of aquatic animals and human pathogenic bacteria - Vibrio riverina

[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), centrifuge at 1000r / min for 10sec after mixing, 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...

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Abstract

The invention provides a gene diagnosis reagent box and its detecting method for aquatic animal (like cyprinoid, Penaeus monodon, Epinephelus awoara, ormer, etc) and human pathogens-Vibrio fluvialis, designed by using a pair of primers designed by the sequence in conservative region of Vibrio fluvialis gene to act as main body. It adopts polyase chain reaction (PCR) technique to qualitatively detect the specific DNA fragments of the Vibrio fluvialis, simply and conveniently, and rapidly, good-specificity, and high-sensitivity; 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, as well as environmental monitoring, avoiding the pathogens infecting and popularizing and it has very great practical value.

Description

technical field [0001] The invention relates to a diagnostic kit and a detection method for aquatic economic animals and human pathogenic bacteria, and mainly relates to a kit and a detection method for gene diagnosis of Vibrio fluvialis. Background technique [0002] Vibrio fluvialis is a halophilic bacterium widely distributed in rivers, bays and coastline areas and in 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 aquatic product morbidity or mass death and human food poisoning caused by Vibrio riverina in various regions of my country. At present, it is known that Vibrio fluvialus can cause many types of aquatic animals such as silver carp, carp, crucian carp, medlar, red carp, bream, etc. The bacteria can infect humans through contaminated aquatic products, c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
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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