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Multiple fluorescence quantitative PCR method for simultaneous and fast detecting three types of pathogenic bacteria in food

A multiple fluorescence quantitative and pathogenic bacteria technology, applied in the direction of microbial measurement/inspection, fluorescence/phosphorescence, biochemical equipment and methods, etc., can solve the problems of lack of positive internal reference, etc., achieve high throughput, good practicability, and operation convenient effect

Inactive Publication Date: 2011-02-16
NANJING INST OF PROD QUALITY INSPECTION
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Problems solved by technology

However, for the three pathogenic bacteria Staphylococcus aureus, Salmonella, and Shigella that must be tested in the national standards of various foods in my country, the multiplex fluorescent quantitative PCR method for simultaneous detection has not yet been reported.
In addition, the current domestic research on the detection of pathogenic bacteria in food by fluorescent quantitative PCR lacks a positive internal reference (IPC) to monitor the reaction system

Method used

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  • Multiple fluorescence quantitative PCR method for simultaneous and fast detecting three types of pathogenic bacteria in food
  • Multiple fluorescence quantitative PCR method for simultaneous and fast detecting three types of pathogenic bacteria in food
  • Multiple fluorescence quantitative PCR method for simultaneous and fast detecting three types of pathogenic bacteria in food

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Embodiment Construction

[0026] 1. Experimental materials, reagents and instruments

[0027] DNA microquantification kit, Invitrogen Company, USA; Taq enzyme and buffer, Takara Company, Japan; media were purchased from Beijing Luqiao; DNA molecular weight standards, lysozyme, proteinase K, etc. were purchased from Nanjing Shengxing; primers and double-labeled Taqman The probes were all synthesized by Shanghai Shenggong Company. Fast fluorescent quantitative PCR instrument, ABI 7500 Fast, American ABI Company; PCR instrument, American ABI Company; high-speed centrifuge, Sigma3-18K, American Sigma Company.

[0028] 2. Simultaneous rapid enrichment of Salmonella, Staphylococcus aureus and Shigella

[0029] Staphylococcus aureus CECT86T, Salmonella S34 and Shigella ATCC8700 each 1×10 1 At the same time, 1 mL of raw milk or 10% pork homogenate was added to prepare artificially contaminated food samples. Prepare the mixed culture medium of Gram-negative bacteria general GN medium and 7.5% NaCl broth vol...

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Abstract

The invention belongs to the technical field of food quality safety detection, and particularly relates to a method in which the processes of fast extraction of co-enrichment and bacterium gene groups and multiple Taqman fluorescence quantitative PCR are used to carry out simultaneous and fast detection on salmonella, staphylococcus aureus and shigella in food and a recombined positive internal reference DNA is designed to monitor PCR reaction so as to guarantee the accuracy of a detected result. The result proves that the method has good specificity for target pathogenic bacteria, the detection limit can reach 101cfu / PCR, and all components of a reaction system do not generate cross interference. Blind sample detection on artificially contaminated foods verifies the practicability of the method. Positive internal reference DNA effectively guarantees the reliability of detection, and avoids the occurrence of false negative results. In conclusion, the invention establishes a multiple fluorescence quantitative PCR method for simultaneous and fast detecting staphylococcus aureus, salmonella and shigella in the food, and provides a new method for high-throughput detection of food origin diseases.

Description

technical field [0001] The invention belongs to the technical field of food quality and safety detection, and in particular relates to a simultaneous rapid detection method for Salmonella, Staphylococcus aureus and Shigella in food by using a multiplex fluorescence quantitative PCR method and an application thereof. Background technique [0002] Staphylococcus aureus, Salmonella and Shigella are three common food-borne pathogens, and they are also three mandatory indicators for pathogen detection in my country's national food hygiene standards. Among them, Staphylococcus aureus is the most common pathogen causing human suppurative infection; Salmonella, as a zoonotic intestinal pathogen, has always been at the forefront of food poisoning cases in the world; Shigella is commonly known as Shigella is the main pathogenic bacteria causing bacillary dysentery in humans. [0003] At present, the detection of the above-mentioned pathogenic bacteria in the national standard mainly ...

Claims

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Application Information

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IPC IPC(8): C12Q1/10C12Q1/68C12Q1/14G01N21/64
CPCY02A50/30
Inventor 杨军张驰凌睿刘新梅周骏贵
Owner NANJING INST OF PROD QUALITY INSPECTION
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