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PCR primer pair for detecting pseudomonas fluorescens capable of producing heat-resistant protease in raw milk and application of PCR primer pair

A technology of Pseudomonas fluorescens and raw milk, applied in the field of Pseudomonas fluorescens detection, can solve the problems of high detection limit, missed detection, poor specificity of PCR primers, etc., and achieve simple determination, strong specificity, and good results Accurate and reliable results

Inactive Publication Date: 2021-08-10
OCEAN UNIV OF CHINA
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Aiming at the above-mentioned problems of poor specificity, high detection limit, and missed detection of existing thermostable protease-producing Pseudomonas fluorescens PCR primers, the present invention provides a PCR primer and a method for detecting Pseudomonas fluorescens in raw milk. its application

Method used

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  • PCR primer pair for detecting pseudomonas fluorescens capable of producing heat-resistant protease in raw milk and application of PCR primer pair
  • PCR primer pair for detecting pseudomonas fluorescens capable of producing heat-resistant protease in raw milk and application of PCR primer pair

Examples

Experimental program
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Effect test

Embodiment 1

[0018] Example 1: Specific detection of thermostable protease-producing Pseudomonas fluorescens in raw milk

[0019] (1) Bioinformatics analysis obtained the conserved region in the amino acid sequence of the thermostable protease AprX encoded by the Pseudomonas fluorescens protease gene aprX. The sequence of the conserved region was input into the PCR primer design software Primer Premier5, and the following degenerate primer sequences were designed (primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd.):

[0020] F3:5'-WSNGGNGGNGAYTTYCAYATGAC-3', (SEQ ID NO:1)

[0021] R3:5'-RTCRTTNCCNCCNCCRTCCC-3', (SEQ ID NO:2)

[0022] (2) Inoculate 4 strains of protease-producing Pseudomonas fluorescens derived from raw milk and 6 common contaminated strains in raw milk into LB medium for recovery for 24 hours, and extract the genome according to the instructions of the genome kit. The extracted genome was used as a template in the PCR amplification system.

[0023] Add...

Embodiment 2

[0028] Example 2: Determination of detection limit of Pseudomonas fluorescens producing thermostable protease in raw milk

[0029] (1) Select a thermostable protease-producing Pseudomonas fluorescens CICC 23250 derived from raw milk and inoculate it in LB medium for recovery for 24 hours. Take 1 mL of the bacterial solution and dilute 6 gradients with a 10-fold concentration gradient dilution method. Take 1 mL of each dilution gradient and extract the genome step by step according to the instructions of the genome kit. At the same time, plate counts were performed on the original bacterial liquid.

[0030] (2) Perform PCR amplification according to the reaction system and reaction conditions in Example 1.

[0031] (3) After the reaction, 5 μL of PCR amplification products were taken for 1.5% agarose gel electrophoresis, and the electrophoresis results were observed after 100V for 30 minutes. figure 2 Middle lanes 2 to 5 (the numbers of Pseudomonas fluorescens were 2.57×10 ...

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Abstract

The invention relates to the technical field of pseudomonas fluorescens detection, and provides a PCR primer for detecting pseudomonas fluorescens in raw milk and application of the PCR primer, so as to solve the problems that the existing PCR primer of pseudomonas fluorescens capable of producing heat-resistant protease is poor in specificity, high in detection limit and missed in detection, wherein an upstream primer F3: 5'-WSNGGNGGNGAYTTYCAYATGAC-3'; a downstream primer R3: 5'-RTCRTTNCCNCCNCCRTCCC-3'. The PCR primer pair has the advantages of being high in specificity, reliable in result and easy and convenient to judge and is suitable for food sample detection, and the detection method is high in speed, accurate and reliable in result, easy and convenient to operate and low in detection cost.

Description

technical field [0001] The invention relates to the technical field of Pseudomonas fluorescens detection, in particular to a pair of PCR primers for detecting Pseudomonas fluorescens producing heat-resistant protease in raw milk and an application thereof. Background technique [0002] Dairy products contain various nutrients needed by the human body and are easily digested and absorbed. In recent years, dairy products have become more and more popular among consumers. Raw milk is the main raw material of various dairy products, and its quality directly affects the quality of dairy products. Pseudomonas fluorescens is a type of microorganism that can grow and reproduce at 0-7°C. It exists widely in all aspects of dairy processing and production, and it is very easy to cause contamination through contact with raw milk. The application of low-temperature cold chain technology in the dairy industry has made Pseudomonas fluorescens with psychrophilic characteristics dominant in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/39
CPCC12Q1/689C12Q1/686
Inventor 易华西乔文君公丕民刘同杰张兰威艾连中王世杰黄冬成
Owner OCEAN UNIV OF CHINA
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