Detecting method for food-borne pathogenic bacteria listeria monocytogenes

A technology for the detection of Listeria monocytogenes and its purpose, which is applied in the field of detection of food-borne pathogenic bacteria Listeria monocytogenes, can solve the problems of long inspection cycle, inability to be widely used and popularity in the market, and achieve simple detection , effectively the occurrence of listeriosis monocytogenes epidemic, and the effect of inhibiting the occurrence of listeriosis monocytogenes epidemic

Active Publication Date: 2016-06-15
NINGBO UNIV +2
View PDF5 Cites 9 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method does not require high experimental equipment, but the inspectio

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
  • Detecting method for food-borne pathogenic bacteria listeria monocytogenes
  • Detecting method for food-borne pathogenic bacteria listeria monocytogenes
  • Detecting method for food-borne pathogenic bacteria listeria monocytogenes

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0024] Example 1: Design and optimization of primers and probes

[0025] Step 1) Design primers based on the hly gene (hlyA) encoding hemolysin O (listeriolysinO) of Listeria monocytogenes:

[0026] The public listeriolysinO encoding gene hly was found through NCBI, and its conserved region is at positions 1657-3060 of the hly gene. After homology analysis, the gene located in 1645-3069 was used as the target fragment for RPA primer design.

[0027] Design 4 sets of primer pairs for optimal primer screening, the primer sets and sequences are as follows:

[0028] Group 1:

[0029] Forward primer F1-RPA: 5′-CCGTAAGTGGGAAATCTGTCTCAGGTGATGTAG-3′ (33bp)

[0030] Reverse primer R1-RPA: 5′-TTGTCTTTTAAGAAGTTTGTTGTATAGGCAATGGG-3′ (35bp)

[0031] Product length: 220bp

[0032] Group 2:

[0033] Forward primer F2-RPA: 5′-CTTTTGACGCTGCCGTAAGTGGGAAATCTGT-3′ (31bp)

[0034] Reverse primer R1-RPA: 5′-TTGTCTTTTAAGAAGTTTGTTGTATAGGCAATGGG-3′ (35bp)

[0035] Product length: 230bp

[0036] Group 3:

[0037] Forwar...

Example Embodiment

[0053] Example 2: Detection of the effects of primers and probes

[0054] Establishment of isothermal amplification method of recombinant enzyme polymerase

[0055] Include the following steps

[0056] 1) Preparation of RPA reaction system:

[0057] The final concentration of each primer is: primer F-RPA and primer R-RPA each 420nmol / μL; probe P120nmol / μL; buffer composition and concentration: Tris-HCl (pH7.9) 50mmol / μL, KAc (potassium acetate ) 100mmol / μL, DTT (dithiothreitol) 2mmol / μL, 5% PEG20mol / μL, dNTPs200umol / μL, ATP3mmol / μL, pcr (phosphocreatine kinase) 50mmol / μL, CK (creatine kinase) 100ng / μL, DNA polymerase Bsu30ng / uL, single-stranded binding protein Gp32300ng / uL, recombinase UvsX240ng / uL, auxiliary enzyme UvsY60ng / uL, endonuclease Nfo200ng / uL; sample DNA template 10ng, add double distilled water to make no reaction The volume of the reaction mix is ​​47.5μL; 280mmol / μLMgAc (magnesium acetate) is 2.5μL, so that the total volume of the reaction system is 50μL. Magnesium a...

Example Embodiment

[0068] Example 3: Application of RPA-LFD to artificially contaminated samples

[0069] 3.1 Materials: pork (P), chicken (C), fish (B), beef (F); Mengniu pure milk (M). The materials were purchased from supermarkets.

[0070] 3.2 Reagents: Brain Heart Infusion (BrainHeart Infusion, BHI) sterile liquid medium, sterile water (nuclease-free)

[0071] 3.3 Sample processing:

[0072] 3.3.1 Meat

[0073] After skin and bone removal, wash with sterile water, cut into micro-pieces, weigh 50-100mg, add 400uL pre-cooled sterile double distilled water, manually grind with a tissue grinder to make a homogenate. Centrifuge at 4000rpm for 15min, discard the upper fat layer and the lower sediment, take the middle layer, pass the 0.22um membrane, and sterilize.

[0074] 3.3.2 Milk

[0075] Take an appropriate amount of milk, dilute it 50 times with sterile double distilled water, lyse it directly, pass it through a 0.22um film, and sterilize.

[0076] 3.4 Sample preparation

[0077] 3.4.1 Negative control...

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 provides a detecting method for food-borne pathogenic bacteria listeria monocytogenes. The listeria monocytogenes is safely, specially, rapidly, sensitively and easily detected on site, and the defects of the existing traditional detection technology are overcome. The method is particularly suitable for on-site detection and can effectively restrain occurrence of the listeria monocytogenes epidemic situation in time, and the food safety guarantee system is perfected. The sequence of an adopted forward primer is shown in SEQ ID NO:1, the sequence of an adopted reverse primer is shown in SEQ ID NO:2, and the sequence of a probe is shown in SEQ ID NO:3.

Description

technical field [0001] The invention belongs to the technical field of microorganism detection, and in particular relates to a method for detecting food-borne pathogenic bacteria Listeria monocytogenes. Background technique [0002] Listeria monocytogenes (Listeriamonocytogenes, LM) is Bacteria (Bacteria), Firmicutes (Firmicutes), Bacilli (Bacilli), Bacillales (Bacillales), Listeria (Listeria), for Brevibacterium positive, is the pathogenic bacteria of zoonosis, and is called one of "four major food-borne pathogenic bacteria" by the World Health Organization (WHO). It is widely distributed in nature, such as meat, vegetables, dairy products, and aquatic products. It can be detected in meat, vegetables, dairy products, and aquatic products, and because it can form biofilms, it has strong adaptability and can cause pollution during food production, processing and storage. After the poor, pregnant women, newborns, and the elderly eat these contaminated foods by mistake, they m...

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
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/01
CPCC12Q1/6844C12Q1/689C12Q2521/507C12Q2565/625
Inventor 朱鹏高威芳黄海龙严小军范建忠
Owner NINGBO UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap