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

Method for rapidly detecting food-borne salmonella based on PagN genes

A Salmonella, food-borne technology, applied in the direction of biochemical equipment and methods, microbiological measurement/inspection, resistance to vector-borne diseases, etc., can solve the problems of low detection limit, long time, etc., to ensure the effectiveness and Accuracy, intuitive result judgment, and easy promotion effect

Pending Publication Date: 2020-03-20
TIANJIN UNIV
View PDF1 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a method for rapid detection of food-borne Salmonella based on the PagN gene, which solves the problems of low detection limit, long time and making up for the lack of single detection technology in the prior art

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 rapidly detecting food-borne salmonella based on PagN genes
  • Method for rapidly detecting food-borne salmonella based on PagN genes
  • Method for rapidly detecting food-borne salmonella based on PagN genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: the establishment of detection method

[0032] Step 1, search Salmonella PagN virulence gene according to Genbank, remove its transmembrane domain and design primers, the upstream primer is: 5'-AAAGAAGGGGCCTATATCACCG-3' (SEQ ID No.1), the downstream primer is: 5'-TTAAAATGCGTAAGTGATGCC-3' (SEQ ID No.2), the genomic DNA of different serotypes of Salmonella was extracted, and the results of PCR amplification showed that the virulence gene exists in most of the Salmonella, with a broad spectrum and a single amplified band, which is 657bp in length of DNA fragments.

[0033] PCR amplification reaction conditions: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 sec, annealing at 52°C for 30 sec, extension at 72°C for 1 min, 30 cycles of amplification, and finally extension at 72°C for 10 min.

[0034]Step 2: Construct the pET-28a-PagN prokaryotic expression vector, express and purify the prokaryotic recombinant protein, immunize rabbits with 1 m...

Embodiment 2

[0043] Embodiment 2; Immunomagnetic beads capture efficiency and parameter optimization

[0044] To explore the optimal parameters of the experiment of capturing Salmonella with immunomagnetic beads in order to improve the capture efficiency. Multiple conditions such as the amount of antibody coating, coupling time, and the amount of immunomagnetic beads during capture were optimized by controlling variables. First, different volumes of PagN polyclonal antibodies were coupled with 1 mg of magnetic beads to determine the optimal antibody amount. Measure the amount of antibody coupled to the magnetic beads at different time points to explore the optimal coupling time. After the preparation of the immunomagnetic beads, different concentration gradients were set for 10 5 CFU / mL Salmonella bacteria liquid was captured, and after obtaining the optimal amount of immunomagnetic beads, Salmonella with different concentration gradients were captured, and the optimal capture parameters ...

Embodiment 3

[0045] Embodiment 3: Scanning electron microscope observes and captures the situation

[0046] In order to visually determine whether the constructed PagN immunomagnetic beads can capture Salmonella, observation was carried out under the scanning electron microscope. A single colony of Salmonella CMCC 619 was picked and inoculated in 5mL LB liquid medium by streaking on the plate, and cultured overnight on a shaker at 37°C. Centrifuge at 4000rpm for 5min, wash with 1mL sterile double distilled water and dilute to 10 4 -10 5 CFU / mL, according to the method described in Example 1, 0.2 mg of immunomagnetic beads were added for capture. After capture, the magnetic frame adsorbed the immunomagnetic beads-bacteria complex, and washed 3 times with 1 mL of sterile double distilled water. The washed complex was resuspended in 200 μL sterile double-distilled water, the glass slide was cut into 1 cm squares, 20 μL of the complex was dropped onto the coverslip, fixed, and air-dried in an...

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 rapidly detecting food-borne salmonella based on PagN genes. Polyclonal antibodies prepared by utilizing salmonella conservative virulence genes PagN and carboxylated magnetic beads are coupled to construct immunomagnetic beads, and the immunomagnetic beads are used with a fluorescent quantitative PCR detection method, so that the problems of complexity, time consumption and the like in a traditional culture method are avoided. The method is rapid and effectively and economical in cost, is real-time and visible, and can be used for implementing specific detection and detection of the salmonella in quantitative food.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to the detection of food-borne pathogenic bacteria-Salmonella. Background technique [0002] Salmonella sp. is a Gram-negative bacillus that can cause zoonoses and parasitizes animals and humans with similar biochemical reactions and antigenic structures. It belongs to the family Enterobacteriaceae. Salmon was isolated during the cholera epidemic in 1885. Salmonella choleraesuis, so far there are six intestinal subtypes of Salmonella, more than 2,600 intestinal serotypes have been found, the most common serotypes are Salmonella enterica, Salmonella enteritidis, Bacillus choleraesuis, etc., diet such as pork, milk etc. are the common ways to infect Salmonella. The food infected with Salmonella enters the body through the oral cavity after oral administration, and then enters the blood through the lymphatic system. Infection, the main infection symptoms are persistent fever, diarrhea, vomit...

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): C12Q1/6804C12Q1/06C12Q1/10
CPCC12Q1/6804C12Q2531/113C12Q2563/107C12Q2563/143C12Q2563/149Y02A50/30
Inventor 黄金海华德平付雅莉张蕾
Owner TIANJIN 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