Pseudomonas fluorescens LAMP (loop-mediated isothermal amplification) detection agent and kit

A Pseudomonas fluorescens detection reagent technology, applied in the field of Pseudomonas fluorescens LAMP detection reagents and kits, to achieve high amplification efficiency, simple operation, and short detection time

Inactive Publication Date: 2012-03-21
默沙东(宁波)动物保健科技有限公司
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Problems solved by technology

At present, there is no research to establish specific, sensitive and easy-to-operate Pseudomonas fluorescens LAMP detection reagents and kits

Method used

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Embodiment Construction

[0017] Below in conjunction with embodiment the present invention is described in further detail.

[0018]1. Design primers: select the cDNA of Pseudomonas fluorescens as the target, and use PRIMEREXPLORER V4 software to design primers (including the five F3, B3, Y-LB, FIP, and BIP). Bioengineering Limited completed. Compare the primers with the target, perform routine screening tests and analysis, and obtain specific, sensitive, and stable 5 primers for the above-mentioned F3, B3, Y-LB, FIP, and BIP; After optimizing the experiment, determine the reaction conditions with the best amplification efficiency and specificity.

[0019] 2. Reaction temperature optimization: Design a gradient LAMP reaction program, the reaction temperatures are: 61°C, 63°C, 65°C, and 63°C is optimized as the best reaction temperature among these temperatures.

[0020] 3. Suitable Mg 2+ Concentration selection: Mg in the reaction system 2+ (2.5 mM) was added in the following order: 0 μL, 2...

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Abstract

The invention discloses a pseudomonas fluorescens LAMP (loop-mediated isothermal amplification) detection agent and kit. The pseudomonas fluorescens LAMP detection agent is characterized by constructing five primers having good amplification efficiency and specificity: F3:GCGCGAATACTTCAAGTCCA, B3:GTACAGCTGCGAAGTCTGC, Y-LB:TCAACCACGGGGAGCAT, FIP:AGGTGGGGTTGCTGCTGTAGAGGTCAACCTGCTGTATGTGA and BIP:TGCCCAAGACCATCCTTTCCAAACAGTTCGTTGGTGTCCG. Compared with the conventional PCR (polymerase chain reaction), the pseudomonas fluorescens LAMP detection reagent and kit can overcome the disadvantages of high time consumption, high requirements for equipment and the like. The agent and kit composed of the five specific primers can carry out quick and accurate qualitative detection on pseudomonas fluorescens in a sample, and have the advantages of good specificity, simple operation process, visual result, low cost and the like.

Description

technical field [0001] The invention relates to the detection technology of Pseudomonas fluorescens, in particular to a detection reagent and kit for Pseudomonas fluorescens LAMP. Background technique [0002] Pseudomonas fluorescens ( Pseudomonas fluorescens Migula) belongs to the genus Pseudomonas, and it reproduces quickly at 4°C. It is one of the microorganisms that are most likely to pollute and cause corruption and even human diseases in the production, storage and sales of aquatic products. dominant bacteria. The detection methods of Pseudomonas mainly include serological detection (DAS-ELISA), reverse transcription-polymerase chain reaction (PCR) technology and loop-mediated isothermal amplification technology (LAMP). For example, the invention patent with the notification number CN101403001 discloses a kit consisting of reaction solution, Bst DNA polymerase, stabilization solution, sample pretreatment solution, chromogenic solution and positive control solution t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 苏秀榕应琪周君李成华张春丹李晔
Owner 默沙东(宁波)动物保健科技有限公司
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