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Real-time fluorescence PCR based method for rapid screening of pathogenic microorganisms in cucumber

A real-time fluorescent, golden-yellow technology, applied in the field of food microbial identification and application, can solve problems such as hidden dangers of food safety, outflow of juice and nutrients, pollution, etc., and achieve the effect of rapid process, increased sample size, and improved detection efficiency.

Inactive Publication Date: 2016-10-12
TIANJIN INSTITUE OF QUALITY STANDARD & TESTING OF AGRICULTUAL PRODS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The surface of cucumbers may be polluted by various microorganisms during the process of planting, harvesting, storage, transportation and sales, and if the integrity is damaged during the whole process, it is easy to cause the juice nutrients to leak out, which undoubtedly increases the risk of contamination. chance of microbial infection
Therefore, although there have been no cases of collective safety incidents caused by eating fresh fruits and vegetables in China, their potential food safety hazards are relatively large, so prevention and control are particularly important

Method used

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  • Real-time fluorescence PCR based method for rapid screening of pathogenic microorganisms in cucumber
  • Real-time fluorescence PCR based method for rapid screening of pathogenic microorganisms in cucumber
  • Real-time fluorescence PCR based method for rapid screening of pathogenic microorganisms in cucumber

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Embodiment 1

[0044] Taking common green cucumber as a sample, the method of the present invention is used to quickly screen four kinds of food-borne pathogenic bacteria. The preparation method is as follows:

[0045] (1) Take 25g of cucumber samples, and carry out selective enrichment according to the national standard GB 4789 detection method for each pathogenic bacteria;

[0046] (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.

[0047] Real-time fluorescent PCR detection:

[0048] 1. Perform real-time fluorescent...

Embodiment 2

[0067] Taking dry cucumber as a sample, four food-borne pathogenic bacteria were quickly screened by the method of the present invention. The preparation method is as follows:

[0068] (1) Take 25g of cucumber samples, and carry out selective enrichment according to the national standard GB 4789 detection method for each pathogenic bacteria;

[0069] (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.

[0070] Real-time fluorescent PCR detection:

[0071] 1. Perform real-time fluorescent PCR amplification ...

Embodiment 3

[0090] Taking Dutch cucumber as a sample, four food-borne pathogenic bacteria were quickly screened by the method of the present invention. The preparation method is as follows:

[0091] (1) Take 25g of cucumber samples, and carry out selective enrichment according to the national standard GB 4789 detection method for each pathogenic bacteria;

[0092] (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.

[0093] Real-time fluorescent PCR detection:

[0094] 1. Perform real-time fluorescent PCR amplificatio...

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Abstract

The invention discloses a real-time fluorescence PCR based method for rapid screening of pathogenic microorganisms in cucumber. The method combines the traditional bacterial culture method, uses water-boiling method to extract salmonella, staphylococcus aureus, Escherichia coli O157:H7 and Listeria monocytogenes in selectively enriched cucumber, and applies real-time fluorescence PCR technology to screen the four food-borne pathogenic bacteria quickly. The DNA extraction technique adopted by the invention can complete the whole process of sample DNA extraction, and the real-time fluorescence PCR technique can whole process identification of a sample within 90min, therefore the method provided by the invention has the advantages of high efficiency, quickness, real-timeness, simple operation and the like, and provides technical support for rapid screening of food-borne pathogenic bacteria in fresh fruit and vegetable cucumber.

Description

technical field [0001] The invention belongs to the technical field of food microorganism identification and application, and specifically relates to the DNA extraction and real-time analysis of four food-borne pathogenic bacteria, Salmonella, Staphylococcus aureus, Escherichia coli O157:H7 and Listeria monocytogenes in fresh fruits and vegetables cucumbers. Fluorescence rapid screening method. Background technique [0002] Ready-to-eat fresh vegetables and fruits are one of the main carriers for the transmission of foodborne pathogens. Ready-to-eat fresh fruits and vegetables are low-acid foods with high water content and various nutrients that are not resistant to high temperatures. Therefore, consumers often do not cook when eating, but simply wash and eat directly, which is very easy to attract pathogenic microorganisms Infection, there is a risk of food safety. Foodborne pathogenic bacteria that have been reported to contaminate ready-to-eat fruits and vegetables incl...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/14C12Q1/10C12Q1/04C12N15/11
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143C12Q2563/107C12Q2561/113Y02A50/30
Inventor 赵新兰青阔王永陈锐朱珠刘娜王成
Owner TIANJIN INSTITUE OF QUALITY STANDARD & TESTING OF AGRICULTUAL PRODS
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