Method for detecting pseudomonas fluorescens and kit and primers thereof

A technology of Pseudomonas fluorescens and a kit, which is applied in the field of microbial detection, can solve the problems of high false positives and false negatives, long detection time, complicated operation, etc. Effect

Active Publication Date: 2016-03-09
BRIGHT DAIRY & FOOD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The technical problem to be solved in the present invention is to overcome the complex operation of the detection of Pseudomonas fluorescens in the prior art, the detection time is long, and the defects

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  • Method for detecting pseudomonas fluorescens and kit and primers thereof
  • Method for detecting pseudomonas fluorescens and kit and primers thereof
  • Method for detecting pseudomonas fluorescens and kit and primers thereof

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Example Embodiment

[0030] Example 1 Double PCR detection of Pseudomonas fluorescens strains

[0031] (1) Primer synthesis

[0032] Synthesize primers capable of PCR amplification of Pseudomonas fluorescens gyrase (gyrase) B subunit gene and metalloprotease operon (apr) specific gene sequence, the sequence is as follows:

[0033] SEN1-L: 5'-GCCAGCAGAGTCACCTTCCA-3' (SEQIDNO:1),

[0034] SEN2-R: 5'-AGCACAAAGTCGCCACCACC-3' (SEQ IDNO: 2),

[0035] SEN3-L: 5'-TAYGGBTTCAAYTCCAAYAC-3' (SEQIDNO: 3),

[0036] SEN4-R: 5'-VGCGATSGAMACRTTRCC-3' (SEQ ID NO: 4).

[0037] SEN3-L and SEN4-R are degenerate primers, Y, B, V, M, S and R are all degenerate bases, where Y stands for C or T, B stands for G, C or T, and V stands for A, G Or C, M represents A or C, S represents G or C, R represents A or G.

[0038] (2) Reaction system and reaction parameters of the double PCR detection method

[0039] Using the above primers, using the genomic DNA of the Pseudomonas fluorescens strain as a template, the PCR reaction system and react...

Example Embodiment

[0062] Example 2 Detection test of artificial contamination of Pseudomonas fluorescens model strain AS1.867

[0063] The activated Pseudomonas fluorescens (AS1.867) broth was connected to the LB medium and cultured on a shaker to the stable stage. The plate counted to obtain an initial bacterial concentration of 1.8×10 9 cfu / mL. Add 25mLUHT milk sample that has been sterilized by ultra-high temperature (135℃, 3-5s) into 225mL selective enrichment solution (selective enrichment solution includes the following components: peptone 10g, tryptone 10g, yeast extract 10g, NaCL10g, distilled water to a constant volume of 1L), respectively connect to the concentration of 180cfu / mL, 18cfu / mL, 1.8cfu / mL Pseudomonas fluorescens 1mL, each concentration to do 3 parallel experiments. Take samples at 0h, 4h, 6h, 8h, 10h, 12h, 24h, and extract genomic DNA by boiling method. Take 2μL of the sample at each time point as a PCR reaction template and add it to the PCR reaction system for double amplif...

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Abstract

The invention discloses a method for detecting pseudomonas fluorescens and a kit and primers thereof. The method includes the following steps of extracting genomic DNA of a to-be-detected sample, conducting a dual PCR reaction through the primers with the nucleotide sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 respectively with the genomic DNA as the template, and detecting existence of single amplification products, at the position 384bp and the position 194bp, in products of the dual PCR reaction products. The nucleotide sequences of the primers are shown. The kit comprises the primers. The method is low in detection time consumption, low in cost and reliable in detection result, and result judgment is easy, the simple, rapid and sensitive method for detecting pseudomonas fluorescens is provided for the field of microbiological detection, and particularly the detection method which is more rapid and more convenient is provided for the field of food safety detection.

Description

technical field [0001] The invention relates to the field of microorganism detection, in particular to a method for detecting Pseudomonas fluorescens and its kit and primers. Background technique [0002] Pseudomonas Fluorescens (Pseudomonas Fluorescens) belongs to the genus Pseudomonas (Pseudomonas) in taxonomy. The cells of the bacteria are straight short rods with a size of 0.7~0.8×2.3~2.8 microns, rounded at both ends, sterile Hairy, non-spore-forming, Gram-staining negative, with several polar flagella, which are motile by flagella. Pseudomonas fluorescens is a eutrophic or facultative anaerobic bacterium that undergoes strict respiratory metabolism with oxygen as the final electron acceptor, but can perform anaerobic respiration with nitrate as an alternative electron acceptor. Its chemotrophs are heterotrophic and do not require organic growth factors. Almost all species cannot grow in acidic environment. Pseudomonas fluorescens does not produce pyocyanase, oxidase...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/39
CPCC12Q1/689C12Q2600/16
Inventor 陈万义刘振民游春苹高琳
Owner BRIGHT DAIRY & FOOD
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