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Method and detection kit for detecting aeromonas sobria scaleless fish pathogenic bacteria

A kit and technology for pathogenic bacteria, applied in the field of detection of pathogenic bacteria in fish, can solve problems such as lack of research reports

Active Publication Date: 2011-04-06
TONGWEI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for the main pathogenic bacteria of channel catfish and largemouth catfish in scaleless fish, Flavobacterium columnarum and Aeromonas temperatus, there is no relevant research report on PCR detection technology in China.

Method used

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  • Method and detection kit for detecting aeromonas sobria scaleless fish pathogenic bacteria
  • Method and detection kit for detecting aeromonas sobria scaleless fish pathogenic bacteria
  • Method and detection kit for detecting aeromonas sobria scaleless fish pathogenic bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0049] Preparation and composition of embodiment 2 kit

[0050] Composition:

[0051] 1. PCR reaction solution 800 μL (10 μL / time, 80 times, containing primers, dNTPs, Mg 2+ , Taq enzyme buffer)

[0052] 2. Taq enzyme 20μL (0.25μL / time, 80 times)

[0053]3. 6% Chelex-100 16mL (DNA extraction solution, 200μL / time, 80 times)

[0054] 4. DNA positive control 160μL (2μL / time, 80 times)

[0055] 5. Sterilized ultrapure water 2mL

[0056] Preparation method: Taq enzyme buffer (final concentration 2×), upstream and downstream primers (final concentration 0.6μM), dNTPs (final concentration 0.4mM), Mg 2+ (final concentration 4mM) were added to centrifuge tubes one by one, and the total volume was made up to 800 μL with sterile ultrapure water. Taq enzyme is a commercial enzyme purchased from Promega and stored at -20°C. See Example 3 for the preparation method of the DNA positive control. Sterilized ultrapure water was prepared using a Millipore water purifier and dispensed aft...

Embodiment 3

[0057] Embodiment 3 detects the diseased fish sample to be tested

[0058] 1) Extract the DNA of the sampled tissue of suspected diseased fish

[0059] Some samples of diseased and suspected diseased grass carp and carp were collected, and the DNA was rapidly extracted using the chelex-100 (Bio-Rad) boiling method. The method is as follows: Take a small piece of diseased tissue and add 200μL Millipore H 2 O, mash, take out the unmasked tissue pieces, centrifuge the remaining turbid solution at 12,000rpm for 1min, discard the supernatant, add 200μL Millipore H 2 O resuspension. After fully mixing, take 50 μL and add it to 200 μL 6% chelex-100, mix evenly, incubate at 56°C for 20 minutes, shake vigorously, incubate at 100°C for 8 minutes, shake vigorously, and centrifuge at 12,000 for 3 minutes, take the supernatant as PCR The template was detected by the established conventional PCR detection system. Conventional PCR amplification was carried out on a Bio-Rad Mycycler gradi...

Embodiment 4

[0081] Example 4: Detection of Mesothermal and Aeromonas in Water

[0082] 1) Acquisition of bacterial DNA: Take samples from aquaculture water, take 1 mL of water samples with a sterile centrifuge tube, centrifuge at 12,000 rpm for 1 min, discard the supernatant, and add 200 μL of Millipore H 2 O resuspension. After fully mixing, take 50 μL and add it to 200 μL 6% chelex-100, mix evenly, incubate at 56°C for 20 minutes, shake vigorously, incubate at 100°C for 8 minutes, shake vigorously, and centrifuge at 12,000 for 3 minutes, take the supernatant as PCR The template was detected by the established conventional PCR detection system. Conventional PCR amplification was carried out on a Bio-Rad Mycycler gradient amplification instrument, and agarose gel electrophoresis combined with gel imaging system was used to detect and analyze the obtained PCR products. The remaining templates were stored at -20°C for re-examination.

[0083] 2) Use the primers prepared in Example 1 to a...

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Abstract

The invention discloses a method and a detection kit for detecting aeromonas sobria scaleless fish pathogenic bacteria. The kit comprises a primer B, which has sequences represented by a sequence SEQ ID No.1 and a sequence SEQ ID No.2, for amplifying a gene from aeromonas sobria. The method of the invention can be used for quickly and accurately detecting if pathogenic bacteria grow in the bodies of scaleless fish or culturing water, so the specific fish disease prevention and control can be realized.

Description

technical field [0001] The invention relates to a detection technology for fish pathogenic bacteria, in particular to a detection method, a detection kit and corresponding primers for scaleless fish pathogenic bacteria Aeromonas temperatus. Background technique [0002] With the high-density breeding of largemouth catfish, channel catfish and other valuable economic scaleless fish, the occurrence of bacterial diseases is becoming more and more serious, especially in the seedling stage. Therefore, only by constantly improving the prevention and treatment methods and conscientiously implementing the policy of "comprehensive prevention and active treatment" can we receive the expected effect of disease prevention. An important basis for disease prevention is to judge whether there are some specific fish pathogenic bacteria in the aquaculture water body or in the fish body, as well as the type and quantity of the existing pathogenic bacteria, so that appropriate methods can be u...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12R1/01
Inventor 黄冠军肖丹刘衍鹏康琦
Owner TONGWEI
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