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LAMP primer set for quickly detecting pseudomonas fluorescens producing thermostable lipase in raw milk and method

A technique for detecting Pseudomonas fluorescens and primers, which is applied in biochemical equipment and methods, recombinant DNA technology, microbe determination/inspection, etc., to achieve fast speed, simple operation and reliable detection results

Inactive Publication Date: 2018-04-20
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no report on the detection of P.fluorescens that produces heat-resistant lipase in raw milk

Method used

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  • LAMP primer set for quickly detecting pseudomonas fluorescens producing thermostable lipase in raw milk and method
  • LAMP primer set for quickly detecting pseudomonas fluorescens producing thermostable lipase in raw milk and method

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1: Specific detection of thermostable lipase-producing Pseudomonas fluorescens in raw milk

[0030] 1) Obtain the conserved region in the lipase gene sequence of Pseudomonas fluorescens through comparative genome and bioinformatics analysis;

[0031] Input the sequence of the conserved region into the primer design software Primer explorer V4 to design primers. The primer sequence is as follows (primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd.):

[0032] F3: 5'-CGAAGCGTGCAGAAGGC-3', (SEQ ID NO: 1)

[0033] B3: 5'-GTGCCGCGAAAACCGA-3', (SEQ ID NO: 2)

[0034] FIP: 5'-CTTTTCGCCGAAGAACGTGCCCTTTTCATCAGCGCCAGCACTCTT-3', (SEQ ID NO: 3)

[0035] BIP: 5'-GCTATACCACGGCCCAGGTCTTTTTGCCGATTTCCTGCAACTG-3', (SEQ ID NO: 4)

[0036] 2) Inoculate 1 lipase-producing Pseudomonas fluorescens strain isolated from raw milk and 6 common contaminated strains in raw milk into LB medium for recovery for 18 hours, and extract the genome according to the instructions ...

Embodiment 2

[0043] Example 2: Sensitivity detection of thermostable lipase-producing Pseudomonas fluorescens in raw milk

[0044] 1) A strain of Pseudomonas fluorescens isolated from raw milk producing thermostable lipase was inoculated into LB for recovery for 18 hours, and 1 mL of bacterial solution was diluted to 7 gradients by 10-fold concentration gradient dilution method, with 3 mL for each dilution gradient The genome was extracted according to the instructions of the genome kit. At the same time, plate counts were performed on the original bacterial liquid.

[0045] 2) Carry out LAMP amplification according to the reaction system and reaction conditions in Example 1.

[0046] 3) After the reaction, add 1 μL of color reagent SYBR Green I to the LAMP reaction system, and observe the reaction result.

[0047] figure 2 Among them, reaction tubes 2-6 (the DNA contents of thermostable lipase-producing Pseudomonas fluorescens were 4.8×10 6 -4.8×10 2 cfu / reaction tube) was green, i....

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Abstract

The invention discloses an LAMP primer set for quickly detecting pseudomonas fluorescens producing thermostable lipase in raw milk and a method and belongs to the field of food safety inspection, andbelongs to the field of food safety detection. By taking a pseudomonas fluorescens lipase gene as a target gene, a set of specific detection primer set and an LAMP detection system and a reaction condition which are suitable for the primer set are designed and screened. A conventional detection method is long in time and consumes both manpower and physical materials. The adopted method is high insensitivity, strong in specificity and short in time; the cost is low, the operation process is simple, and a PCR instrument or an electrophoresis apparatus is not needed in the reaction process; andany instrument is not needed in the result judgment aspect. The shortcomings of the conventional method are overcome to a certain degree. The LAMP primer set and the method have significance on quickly detecting the quality of the raw milk. The adopted method is suitable for bed-layer on-line monitoring and field detection.

Description

technical field [0001] The invention relates to the technical field of food safety detection, in particular to a detection primer set and detection method for Pseudomonas fluorescens producing heat-resistant lipase in raw milk. Background technique [0002] Low-temperature transportation and processing control the growth of mesophilic bacteria contaminated in raw milk, but cannot effectively control the growth of psychrophilic bacteria. Therefore, psychrophilic bacteria become the dominant microorganisms in raw milk during cold chain storage and transportation. Although most psychrophilic bacteria have died during pasteurization and other high-temperature sterilization processes, even if there are residual viable bacteria, it is not enough to endanger the shelf life of dairy products, but psychrophilic bacteria can secrete a large amount of extracellular heat-resistant fats Enzymes, these fats will still leave residues even after high temperature treatment. Residual heat-re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/689C12Q2531/119
Inventor 张兰威辛亮易华西韩雪
Owner OCEAN UNIV OF CHINA
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