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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
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method does not require high experimental equipment, but the inspection cycle is long, so it cannot be widely used and popularized in the market.

Method used

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  • 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

Embodiment 1

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

[0025] Step 1) carry out primer design according to the hly gene (hlyA) of the encoding hemolysin O (listeriolysinO) of Listeria monocytogenes:

[0026] The public listeriolysinO coding gene hly was found through NCBI, and the site of its conserved region was the 1657-3060 position of the hly gene. After homology analysis, RPA primers were designed with the gene located at 1645-3069 as the target fragment.

[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′-TTGTCTTTTTAAGAAGTTTGTTGTATAGGCAATGGG-3′(35bp)

[0031] Product length: 220bp

[0032] Group 2:

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

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

[0035...

Embodiment 2

[0053] Embodiment 2: the detection of primer and probe effect

[0054] Establishment of Isothermal Amplification Method for Recombinase Polymerase

[0055] Include the following steps

[0056] 1) Prepare the 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 are: 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-strand binding protein Gp32300ng / uL, recombinase UvsX240ng / uL, auxiliary enzyme UvsY60ng / uL, endonuclease Nfo200ng / uL; sample DNA template 10ng, add double distilled water to make unreacted The volume of the reaction mix is ​​47.5 μL; 280 mmol / μL MgAc (magnesium acetate) is 2.5 μL, so that the total volume of the reaction system is 50 μL. Magnesium...

Embodiment 3

[0068] Example 3: Application of RPA-LFD to the detection of artificially polluted samples

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

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

[0071] 3.3 Sample handling:

[0072] 3.3.1 Meat

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

[0076] 3.4 Sam...

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PUM

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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

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Application Information

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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
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