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Method for detecting pathogenic bacteria columnar flavobacterium of alepidote fish and detection kit

A Flavobacterium columnar and kit technology, applied in the field of detection of fish pathogenic bacteria, can solve the problem of no research report

Active Publication Date: 2011-04-13
TONGWEI AGRI DEV CO LTD
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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 columnar and Aeromonas temperatus, there is no relevant research report on PCR detection technology in China.

Method used

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  • Method for detecting pathogenic bacteria columnar flavobacterium of alepidote fish and detection kit
  • Method for detecting pathogenic bacteria columnar flavobacterium of alepidote fish and detection kit
  • Method for detecting pathogenic bacteria columnar flavobacterium of alepidote fish and detection kit

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-RadMycycler gradie...

Embodiment 4

[0081] Embodiment 4: the detection of water body sample

[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-RadMycycler 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 amplify the ...

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Abstract

The invention discloses a method for detecting pathogenic bacteria columnar flavobacterium of an alepidote fish and a detection kit. The kit comprises a primer A, the sequence of the primer A is shown as SEQ ID NO:1-2, and a gene from the columnar flavobacterium is amplified. By adopting the method, whether the pathogenic bacteria are contained in the alepidote fish or an aquaculture water body or not can be quickly and accurately detected, and fish diseases are pertinently controlled.

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 Flavobacterium columnar. 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 b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12R1/20
Inventor 肖丹黄冠军刘衍鹏康琦
Owner TONGWEI AGRI DEV CO LTD
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