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Kit and method for detecting fish pathogenic bacteria

The technology of a pathogenic bacteria and kit is applied in the field of kits for detection of fish pathogenic bacteria, which can solve the problems of non-standard strains, non-representative, and inability to explain the accurate detection of Edwardsiella spp., achieving high sensitivity, highly specific effect

Active Publication Date: 2012-03-21
TONGWEI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Liang Wanwen, "Establishment of PCR diagnostic method for channel catfish septicemia", Journal of Dalian Fisheries University, August 2007, Volume 22, No. 4, discloses a method for detecting channel catfish septicemia by PCR method, but the detection target of this method is a A gene (Edwardsiella ictaluri putative protein, eip) to be verified, whether it is a pathogenic gene needs to be further explored, it is not representative, and the test object of this document is the fish disease laboratory of Guangxi Research Institute. Preserved, not a standard strain, it cannot be stated that the method in this document can accurately detect Edwardsiella

Method used

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  • Kit and method for detecting fish pathogenic bacteria
  • Kit and method for detecting fish pathogenic bacteria
  • Kit and method for detecting fish pathogenic bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Primer Design Experiment

[0026] 1. Primer design

[0027] Search the conserved gene sequences of Edwardsiella phosphosrine transaminase from GenBank, and design primers based on these sequences. Among them, according to the coding gene sequence of Edwardsiella serC (gene sequence number is AF110153.1, which is a pathogenic gene), a pair of primers designed are:

[0028] Ei1F(5-CATGATAATACCCGGTGTTGG-3)

[0029] Ei1R(5-GTATTGCTGGGGAACAACTC-3),

[0030] As shown in SEQ ID NO.1 and SEQ ID NO.2, the size of the expected amplified fragment is 124bp. Primers were synthesized by Shanghai Sangon Biological Services Co., Ltd.

[0031] 2. Primer verification

[0032] (1) Template preparation: activating and preparing a bacterial suspension of Edwardsiella catacili ATCC 33202, and extracting genomic DNA. DNA was extracted using Tiangen bacterial genomic DNA extraction kit.

[0033] (2) Amplification: amplify on the Bio-Rad Mycycler gradient amplification instrume...

Embodiment 2

[0040] Specificity and sensitivity detection of embodiment 2PCR detection system

[0041] 1. Experimental method

[0042] (1) Template preparation: use the genomic DNA of Flavobacterium columnar, Aeromonas temperatus, Vibrio river, Pseudomonas fluorescens, Aeromonas caviae, Aeromonas hydrophila, and Edwardsiella spp. as templates for determination The specificity of the method; with the bacterium solution of the Edwardsiella spp. (concentration range is 0.28 × 10 8 cfu / mL-0.28×10 1 cfu / mL) and gradient concentrations of genomic DNA (concentration range from 25ng / μL to 2.5fg / μL) are the sensitivity of the template assay method. DNA was extracted using the bacterial genomic DNA extraction kit provided by Tiangen.

[0043] (2) Amplification: amplify on the Bio-Rad Mycycler gradient amplification instrument with the primer pair designed in Example 1;

[0044] 20μL PCR reaction system (final concentration):

[0045]

[0046]

[0047] The PCR program was: pre-denaturation...

Embodiment 3

[0055] Composition and method of use of embodiment 3 test kit

[0056] 1. The composition of the kit

[0057] (1), PCR reaction solution (10 μ L / time): buffer solution, primers with nucleotide sequences as shown in SEQ ID NO: 1-2, Mg 2+ , dNTPs;

[0058] (2), Taq enzyme (0.25μL / time);

[0059] (3), 6% chelex-100 (DNA extraction solution, 200μL / time);

[0060] (4), sterilized ultrapure water.

[0061] 2. How to use

[0062] (1) Extract the genomic DNA of the sample to be tested;

[0063] (2) Using the genomic DNA extracted from the sample to be tested as a template, use the aforementioned kit to amplify:

[0064] 20μL PCR reaction system (final concentration):

[0065]

[0066]

[0067] The PCR program was: pre-denaturation at 95°C for 4 min; denaturation at 94°C for 15 s, annealing at 56°C for 10 s, extension at 72°C for 15 s, and 38 cycles; incubation at 72°C for 5 min and termination at 4°C.

[0068] (3) 2% agarose gel electrophoresis to detect PCR amplificatio...

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Abstract

The invention provides a kit for detecting fish pathogenic bacteria; the kit comprises a primer with a nucleotide sequence as shown in SEQ ID NO. 1-2, and the primer is used to amplify genes of edwardsiella ictaluri. The invention also provides a method for detecting fish pathogenic bacteria, and an application of the primer shown in SEQ ID NO. 1-2. The kit provided by the invention can rapidly and accurately detect a pathogenic bacterium of ictalurus punctatus - edwardsiella ictaluri, and thus realizes pertinent control and detection of ictalurus punctatus enteric septicemia, and the invention has wide application prospects.

Description

technical field [0001] The invention relates to a kit for detecting fish pathogenic bacteria, which belongs to the field of biotechnology. Background technique [0002] Edwardsiella ictaluri (Edwardsiella ictaluri) was first reported in 1979 as a new pathogenic bacteria cultured in channel catfish (Ictalurus punctatus). In recent years, 85% of the bacteria isolated from diseased channel catfish were Edwardsiella ictaluri. [0003] The traditional method to detect Edwardsiella similarius is the routine isolation and identification of bacteria, but it takes a long time, the procedure is cumbersome and the accuracy is low. In recent years, methods for detecting and diagnosing diseases by PCR are emerging. Liang Wanwen, "Establishment of PCR diagnostic method for channel catfish septicemia", Journal of Dalian Fisheries University, August 2007, Volume 22, No. 4, discloses a method for detecting channel catfish septicemia by PCR method, but the detection target of this method is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/31C12N15/10C12R1/01
Inventor 黄冠军刘天强刘衍鹏李茂
Owner TONGWEI
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