Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Method for identifying life or death of cells of food-borne pathogenic bacteria

A technology for food-borne pathogenic bacteria and pathogenic bacteria, which is applied in the directions of microorganism-based methods, biochemical equipment and methods, and the determination/inspection of microorganisms. Dead cells, etc.

Inactive Publication Date: 2010-09-29
SOUTH CHINA AGRI UNIV
View PDF0 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to detect food-borne pathogenic bacteria according to the existing PCR technology, there are problems that living cells and dead cells cannot be distinguished, and the specificity and accuracy of detection are low. By combining EMA selective permeability and PCR detection technology , to establish a new method for rapid and effective detection and identification of dead and living cells of Vibrio parahaemolyticus and other food-borne pathogens

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for identifying life or death of cells of food-borne pathogenic bacteria
  • Method for identifying life or death of cells of food-borne pathogenic bacteria
  • Method for identifying life or death of cells of food-borne pathogenic bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] (1) EMA treatment of live / dead cells of Vibrio parahaemolyticus at different concentrations

[0032] Take 1 mL of Vibrio parahaemolyticus live / dead cell suspensions at different concentrations and place them in sterile centrifuge tubes, add EMA at a final concentration of 5-50 μg / mL and place them in the dark for 3-10 min, place the centrifuge tubes in In an ice bath with the tube cap opened, expose to a 650W halogen lamp for 5-15 minutes.

[0033] (2) Preparation of DNA template of Vibrio parahaemolyticus after EMA treatment

[0034] Take 1 mL of the EMA-treated bacterial solution in a sterile centrifuge tube, centrifuge at 10,000 r / min for 5 min, discard the supernatant, wash 2 to 3 times with normal saline, add 100 μL of TE to resuspend, bathe in 100°C water for 10 min, and cool in an ice bath for 10 min. Centrifuge at 12000r / min for 5min, and take the supernatant as a PCR template.

[0035] (3) PCR amplification reaction

[0036] Primer synthesis: The primer sequ...

Embodiment 2

[0043] (1) EMA treatment of mixed bacterial suspensions of Vibrio parahaemolyticus with different proportions of viable cells

[0044] Vibrio parahaemolyticus live cell suspension and dead cell suspension (2.0×10 7 CFU / mL) to prepare viable cells containing 100%, 75%, 50%, 25%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0% (both volume percentages) To the bacterial suspension mixed system, add EMA with a final concentration of 5-50 μg / mL and place in the dark for 3-10 minutes. Place the centrifuge tube in an ice bath and open the cap, and expose to a 650W halogen lamp for 5-15 minutes.

[0045] (2) Preparation of DNA template of Vibrio parahaemolyticus after EMA treatment

[0046] Take 1 mL of the EMA-treated bacterial solution in a sterile centrifuge tube, centrifuge at 10,000 r / min for 5 min, discard the supernatant, wash 2 to 3 times with normal saline, add 100 μL of TE to resuspend, bathe in 100°C water for 10 min, and cool in an ice bath for 10 min. Centrifuge at 12000r / min for 5min...

Embodiment 3

[0053] (1) EMA treatment of different concentrations of Shigella live / dead cells

[0054] Take 1 mL of Shigella live / dead cell suspensions at different concentrations and place them in sterile centrifuge tubes, add EMA at a final concentration of 5-50 μg / mL and place them in the dark for 3-10 min, place the centrifuge tubes in In an ice bath with the tube cap opened, expose to a 650W halogen lamp for 5-15 minutes.

[0055] (2) Preparation of Shigella DNA template after EMA treatment

[0056] Take 1 mL of the EMA-treated bacterial solution in a sterile centrifuge tube, centrifuge at 10,000 r / min for 5 min, discard the supernatant, wash 2 to 3 times with normal saline, add 100 μL of TE to resuspend, bathe in 100°C water for 10 min, and cool in an ice bath for 10 min. Centrifuge at 12000r / min for 5min, and take the supernatant as a PCR template.

[0057] (3) PCR amplification reaction

[0058] Primer synthesis: The primer sequences are as follows, and the length of the amplifi...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a method for identifying life or death of cells of food-borne pathogenic bacteria. The identification method comprises the following steps of: adding cell bacteria suspension of the food-borne pathogenic bacteria into EMA with final concentration of 5 to 50 microgram / milliliter and standing the mixture in dark for 3 to 10 minutes; exposing the mixture for 5 to 15 minutes by using a halogen lamp under the condition of ice bath; extracting the DNA of the bacteria solution; performing PCR and electrophoretic analysis; and proving that the cells of the food-borne pathogenic bacteria are living cells when a specific amplification fragment occurs or dead cells when no specific amplification fragment occurs. By the identification method, the life or death of the food-borne pathogenic bacteria, particularly the vibrio parahaemolyticus, can be rapidly detected, false positive result can be prevented in the detection of an actual sample, reliable basis can be provided for the prevention or control of the food-borne diseases and a scientific method and a theoretical basis can be provided for the sanitary inspection of food and the inspection work of the import and export commodities.

Description

technical field [0001] The invention relates to a method for detecting the activity of microbial cells, in particular to a method for identifying the dead and alive cells of food-borne pathogenic bacteria. Background technique [0002] According to the World Health Organization, 70% of the hundreds of millions of food-borne disease patients worldwide are caused by various pathogenic microorganisms contaminating food and drinking water. The statistics of food poisoning occurrence in my country in the past ten years show that microbial food poisoning ranks first among all kinds of food poisoning pathogens, accounting for about 40% of the scale of food poisoning, and the number of people poisoned accounts for more than half of the total number of poisoned people. Salmonella, Staphylococcus aureus, Vibrio parahaemolyticus, Campylobacter jejuni, Escherichia coli, Shigella, Clostridium botulinum, Listeria monocytogenes, etc. are important foodborne pathogens. [0003] Among them,...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12R1/63C12R1/01
Inventor 钟青萍王丽李青孙远明
Owner SOUTH CHINA AGRI UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com