Method for rapidly and quantitatively screening salmonella in strawberry based on microdroplet digital PCR method

A Salmonella, micro-droplet digital technology, applied in the field of food microbial identification and application, can solve the problems of time-consuming and labor-intensive, complicated and cumbersome Salmonella inspection procedures, and achieve the effect of increasing sample size, rapid process, and improving detection efficiency

Inactive Publication Date: 2016-12-21
TIANJIN INSTITUE OF QUALITY STANDARD & TESTING OF AGRICULTUAL PRODS
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The traditional culture method is a commonly used detection method at present, but the detection procedure of Salmonella is complex and tedious, time-consuming and labor-intensive, and the whole process needs at least 4-7 days to obtain a definite diagnosis result

Method used

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  • Method for rapidly and quantitatively screening salmonella in strawberry based on microdroplet digital PCR method
  • Method for rapidly and quantitatively screening salmonella in strawberry based on microdroplet digital PCR method
  • Method for rapidly and quantitatively screening salmonella in strawberry based on microdroplet digital PCR method

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Embodiment 1

[0045] Using the cream strawberry as a sample, the method of the present invention is used for rapid and quantitative screening of Salmonella. The preparation method is as follows:

[0046] (1) Take 25g of cucumber samples, and carry out selective enrichment according to the national standard GB 4789 detection method for each pathogenic bacteria;

[0047](2) Take 1 mL of the enrichment solutions of Salmonella, Staphylococcus aureus, Escherichia coli O157:H7, and Listeria monocytogenes after selective enrichment, put them into 2 mL centrifuge tubes, centrifuge at 13200 rpm for 10 min, and discard the supernatant; Dissolve in 100 μL of deionized water, seal with a parafilm, and bathe in boiling water for 10 minutes; take it out and quickly freeze it in a -20°C refrigerator for 10 minutes; remove the parafilm, centrifuge at 13,200 rpm for 3 minutes, and the supernatant is DNA.

[0048] Real-time fluorescent PCR detection:

[0049] 1. Perform real-time fluorescent PCR amplificat...

Embodiment 2

[0060] Using the Charlie strawberry as a sample, the method of the present invention is used for rapid and quantitative screening of Salmonella. The preparation method is as follows:

[0061] (1) Take 25g of cucumber samples, and carry out selective enrichment according to the national standard GB 4789 detection method for each pathogenic bacteria;

[0062] (2) Take 1 mL of the enrichment solutions of Salmonella, Staphylococcus aureus, Escherichia coli O157:H7, and Listeria monocytogenes after selective enrichment, put them into 2 mL centrifuge tubes, centrifuge at 13200 rpm for 10 min, and discard the supernatant; Dissolve in 100 μL of deionized water, seal with a parafilm, and bathe in boiling water for 10 minutes; take it out and quickly freeze it in a -20°C refrigerator for 10 minutes; remove the parafilm, centrifuge at 13,200 rpm for 3 minutes, and the supernatant is DNA.

[0063] Real-time fluorescent PCR detection:

[0064] 1. Perform real-time fluorescent PCR amplifi...

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Abstract

The invention discloses a method for rapidly and quantitatively screening salmonella in the strawberry based on a microdroplet digital PCR method. According to the method, salmonella invA virulence genes are adopted for synthesizing specific primers and probes, the bacterial DNA in the strawberry is extracted through a Chelex-100 method, and the salmonella is rapidly and quantitatively screened by adopting the microdroplet digital PCR technology. According to the established method, plasmids and a standard curve do not need to be constructed, so that the problem that the value determining accuracy of the sample is influenced by the quantity value deviation of the standard curve is avoided; in the whole detection process, the whole-process identification for the sample can be completed within 4h, therefore, the method has the advantages of being efficient, rapid, real-time, easy and convenient to operate, and a technical support is provided for rapidly and quantitatively screening the salmonella in the fresh fruits and vegetables, such as the strawberry.

Description

technical field [0001] The invention belongs to the technical field of food microorganism identification and application, and in particular relates to a DNA extraction method for Salmonella and strawberry in fresh fruits and vegetables and a rapid quantitative screening method for microdroplet digital PCR. Background technique [0002] Ready-to-eat fresh vegetables and fruits are one of the main carriers for the transmission of foodborne pathogens. At present, the General Administration of Quality Supervision, Inspection and Quarantine has listed the risk monitoring of food safety as a major solution to people's livelihood. There are many factors that affect food safety, and food safety problems caused by microbial hazards account for 40% of the total. [1-2] . At present, China is facing severe challenges in terms of food-borne pathogenic microorganisms, and it is urgent to make a reasonable risk assessment of potential hazards, and turn passive into proactive to control th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/06C12N15/11C12R1/42
CPCC12Q1/689C12Q1/6851C12Q2563/159C12Q2561/113C12Q2563/107
Inventor 赵新王永兰青阔陈锐朱珠刘娜王成
Owner TIANJIN INSTITUE OF QUALITY STANDARD & TESTING OF AGRICULTUAL PRODS
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