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Multiple PCR detection kit for rapidly identifying pathogenic Pseudomonas plecoglossicida, and method thereof

A technology of Pseudomonas ayumi and Pseudomonas, applied in the direction of microorganism-based methods, microorganism measurement/inspection, biochemical equipment and methods, etc., can solve the lack of specificity and the inability to distinguish virulence pathogens at the same time Strains and non-virulent environmental isolates, etc., to achieve the effect of short detection time, simple equipment requirements, and simple operation

Inactive Publication Date: 2014-08-20
ZHEJIANG WANLI UNIV
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  • Summary
  • Abstract
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Problems solved by technology

The detection of fish pathogenic bacteria Pseudomonas ayumi is like a Chinese invention patent application with application number 201210466878.5 (published number is CN102952884A) "A LAMP detection kit and detection of fish pathogenic bacteria Pseudomonas ayumi Method" discloses the following kit: including reaction buffer, MgSO4 solution, dNTP solution, Bst DNA polymerase solution, betaine solution, FIP / BIP primer solution, F3 / B3 primer solution and double distilled water, the LAMP detection reagent The cassette has high sensitivity and specificity to Pseudomonas ayumi, but this method uses the ubiquitous housekeeping gene rpoD in the strain as the target gene sequence, and cannot distinguish between virulent pathogenic strains and avirulent environmental isolates at the same time , lacking sufficient specificity

Method used

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  • Multiple PCR detection kit for rapidly identifying pathogenic Pseudomonas plecoglossicida, and method thereof
  • Multiple PCR detection kit for rapidly identifying pathogenic Pseudomonas plecoglossicida, and method thereof
  • Multiple PCR detection kit for rapidly identifying pathogenic Pseudomonas plecoglossicida, and method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0034] A pathogenic Ayu Pseudomonas multiplex PCR detection kit, the kit is specifically composed of the following reagents:

[0035] 0.05 mL of Taq DNA polymerase, 0.5 mL of 10× PCR buffer, 0.25 mL of deoxyribonucleoside triphosphate mixture, 5 mL of redistilled water, 200 μL of 3 pairs of specific primers with a concentration of 10 μM, respectively, Pseudomonas RNA polymerase Subunit gyrB upstream and downstream primers P1, P2, Ayu Pseudomonas gyrB upstream and downstream primers P3, P4 and pathogenic Ayu Pseudomonas type III secretion system regulator exsA upstream and downstream primers P5 and P6.

[0036]Among them, relatively degenerate primers were designed based on the internal conserved sequences of the gyrB genes of Pseudomonas fluorescens, Pseudomonas putida and Pseudomonas ayumi with gene accession numbers CP000094.2, KC189956.1, and AB178859.1 , the target sequence length is 745bp; among them, the upstream and downstream primers of Pseudomonas RNA polymerase subun...

Embodiment 3

[0053] According to the method of Example 2, the DNA template solution extracted in the method was serially diluted in multiples, with a concentration ranging from 1050 ng / μL to 1 ng / μL, and the same multiplex PCR detection as in Example 2 was performed, and it was clear within the concentration range of ≥4 ng / μL 3 specific purpose bands were amplified accurately, showing that the sensitivity of DNA that can be detected by the multiplex PCR established by the present invention is 4ng / μL, and the results are as follows: figure 2 As shown, where M is the DNA molecular weight standard DL2000, 1-10 are template DNA concentrations of 1050ng / μL, 525ng / μL, 258ng / μL, 129ng / μL, 65ng / μL, 32ng / μL, 16ng / μL, 8ng / μL, respectively. Multiplex PCR detection results at μL, 4ng / μL, 2ng / μL, 1ng / μL, lane 11 is negative control.

Embodiment 4

[0055] Respectively for Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas ayumi (pathogenic strains and environmental isolates, the environmental isolates are provided by the Marine Microorganism Collection Center of the State Oceanic Administration), Vibrio harveyi, Vibrio parahaemolyticus, Vibrio alginolyticus and Aeromonas hydrophila etc. were extracted template DNA according to the method in Example 2, and carried out the same multiple PCR detection, and the electrophoresis results were as follows: image 3 , indicating that the multiplex PCR established by the present invention can specifically detect Pseudomonas ayumi, and at the same time distinguish pathogenic strains from environmental isolates. Among them, M is the DNA molecular weight standard DL2000, and lanes 1-9 represent Pseudomonas ayuyui NB2011, environmental isolates of Pseudomonas ayumi (MCCC1A00022), Pseudomonas putida (CGMCC1.8092), Pseudomonas fluorescens bacterium (CGMCC1.6...

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Abstract

The present invention discloses a multiple PCR detection kit for rapidly identifying pathogenic Pseudomonas plecoglossicida, and a method thereof. The kit comprises TaqDNA polymerase, a PCR buffer, a deoxyribonucleoside triphosphate mixture, double distilled water, and primer pairs of Pseudomonadaceae, Pseudomonas plecoglossicida and pathogenic Pseudomonas plecoglossicida. The multiple PCR detection method for rapidly identifying pathogenic Pseudomonas plecoglossicida by using the kit comprises: primary separation of Pseudomonadaceae, and multiple PCR amplifications. Compared with the kit and the method in the prior art, the kit and the method of the present invention have the following advantages that: with application of the kit of the present invention to detect, Pseudomonadaceae, Pseudomonas plecoglossicida and pathogenic Pseudomonas plecoglossicida corresponding to the DNA primer pairs of the three Pseudomonadaceae can be rapidly detected, and with the detection method of the present invention, the presence of the Pseudomonadaceae can be rapidly, sensitively and specifically determined by using the kit, and the pathogenic stain and the non-virulent environment isolation strain can be distinguished.

Description

technical field [0001] The invention relates to a detection technology of Pseudomonas ayu, in particular to a multiplex PCR detection kit and a method for rapidly identifying pathogenic Pseudomonas ayu. Background technique [0002] Pseudomonas species that have been reported to be pathogenic to fish include Pseudomonas anguillacida, P. fluorescens, P. putida, and sweet fish Pseudomonas (P. plecoglossicida), etc., the latter three belong to the DNA type I of Pseudomonas fluorescens, and are similar in morphology and some physicochemical properties, especially Pseudomonas putida and Pseudomonas ayu, These two bacteria belong to the Pseudomonas putida group in terms of taxonomy, and are very similar in physical and chemical properties. The 16S rRNA gene sequence homology is over 99%. Conventional physical and chemical property identification and 16S rRNA gene sequencing comparison are not effective. Distinguish the two bacteria. Pseudomonas ayu NB2011 is a virulent strain th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/38
CPCC12Q1/686C12Q2537/143
Inventor 毛芝娟李梅芳陈吉刚
Owner ZHEJIANG WANLI UNIV
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