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A detection method and detection kit for pathogenic Listeria monocytogenes

A technology of Listeria monocytogenes and detection kits, which can be applied in measurement devices, instruments, scientific instruments, etc., can solve the problems of indistinguishable, increased operation steps, and limited wide application, and achieves a rapid popularization and use, rapid response. Effect

Active Publication Date: 2019-12-20
GUANGDONG INST OF ECO ENVIRONMENT & SOIL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The common detection of Listeria monocytogenes mainly relies on PCR technology. Although the sensitivity is high, it requires accurate temperature amplification, expensive detection equipment, and the need to break up the detection bacteria. In addition, PCR detection cannot distinguish single cells in the sample. The number of viable and dead L. monocytogenes severely limits their widespread use
It is also reported that ELISA technology is used to detect Listeria monocytogenes, but it often needs to introduce enzymes for signal output, involving multiple washing and separation processes, and increasing the operation steps

Method used

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  • A detection method and detection kit for pathogenic Listeria monocytogenes
  • A detection method and detection kit for pathogenic Listeria monocytogenes
  • A detection method and detection kit for pathogenic Listeria monocytogenes

Examples

Experimental program
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Effect test

Embodiment 1

[0046] A detection kit for pathogenic bacterium Listeria monocytogenes, comprising the following components:

[0047] (1) Nucleic acid DNA1, DNA2, the sequence is as follows:

[0048] DNA1: 5'-NH 2-AAAAAA(a)-CTGCACA(b)-GGGTAGGG(c)-3' (SEQ ID NO: 2);

[0049] DNA2: 5'-CGGGTTGGG(e)-TCTGCTG(b*)-AAAAAA(d)-NH 2 -3' (SEQ ID NO: 3).

[0050] (2) Listeria monocytogenes antibody;

[0051] (3) Listeria monocytogenes standard solution;

[0052] (4) PBS buffer, pH7.4, containing 200mM NaCl and 50mM KCl;

[0053] (5) hemin solution;

[0054] (6) Chromogenic buffer, containing 26.6mM citric acid, 51.4mM disodium hydrogen phosphate, 25mM KCl, 10μL 0.5% TMB, 20μL 30% H 2 o 2 , pH=5.0.

Embodiment 2

[0056] A detection method for pathogenic bacteria Listeria monocytogenes is carried out according to the following steps:

[0057] (1) The Listeria monocytogenes antibody is linked to the amino-modified nucleic acid DNA1 and DNA2: 6mg / mL antibody is fully dissolved in PBS buffer (pH7.4, containing 200mM NaCl and 50mM KCl), and then add 20mM EDC and 5mM NHS ( EDC and NHS react to form an amide bond, so that the amino-modified nucleic acid can be linked to the antibody), mix well, and react at 4°C for 2 hours, then add 5M amino-modified DNA1 and DNA2, mix well, and react at 4°C for 12 hours , forming antibody-nucleic acid complexes.

[0058] (2) In step (1), add live Listeria monocytogenes solution or solution to be tested in different concentrations, mix thoroughly, and react at room temperature for 40 minutes.

[0059] (3) Add 0.5M hemin solution (hemin), mix thoroughly, and react at room temperature for 30 minutes.

[0060] (4) Take out the 50L reaction solution in step (3)...

Embodiment 3

[0062] Detection of different concentrations of Listeria monocytogenes:

[0063] Prepare Listeria monocytogenes (live bacteria) standard solution, the number of bacteria is 1×10 3 CFU / mL, 1x10 4 CFU / mL, 1x10 5 CFU / mL, 1x10 6 CFU / mL, 1x10 7 CFU / mL, 1x10 8 CFU / mL.

[0064] Different Listeria monocytogenes solutions were added to the reaction system described in Example 2 respectively, and the experimental results were observed after sufficient reaction, as figure 2 As shown, 1x10 3 CFU / mL of Listeria monocytogenes can produce a clear blue change, indicating that its detection limit is 1x10 3 CFU / mL. As the concentration of L. monocytogenes increases, the color also increases and gradually becomes saturated.

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Abstract

The invention discloses a detection method and a detection kit for pathogenic listeria monocytogenes, belonging to the field of detection on pathogenic bacteria. An antibody is connected with DNA to detect listeria monocytogenes, and distance between DNA sequences is shortened after the antibody captures the listeria monocytogenes, so that G-tetramer sequences divided into two parts get close, anda G-tetramer structure with catalytic activity is formed; the formed G-tetramer has catalytic activity similar to that of horseradish peroxidase, so as to catalyze a colorless substrate to turn blue;and the concentration of the listeria monocytogenes is inpositive correlation with blue variation, so that the concentration of the listeria monocytogenes in a detection system can be judged. The detection device and detection kit are high in sensitivity and good in specificity, can directly realize detection for viable bacteria, and do not need a crushing and separating process. According to theinvention, no detection instrument is needed in the detection process, the result can be seen directly by naked eyes, and the detection method and the detection kit have the advantages of being simple in operation, low in cost and rapid in response, and can be used for quickly detecting the content of the listeria monocytogenes in a clinical sample, an environment and food.

Description

technical field [0001] The invention belongs to the field of detection of pathogenic bacteria, and in particular relates to a detection method and a detection kit of pathogenic Listeria monocytogenes. Background technique [0002] Listeria monocytogenes (Listeria monocytogenes), referred to as Listeria monocytogenes, is a zoonotic pathogen. It can cause listeriosis in humans and animals, and the main manifestations after infection are sepsis, meningitis and mononucleosis. It exists widely in nature. Listeria monocytogenes in food is dangerous to human safety. The bacteria can still grow and reproduce in an environment of 4°C. It is one of the main pathogenic bacteria that threaten human health in refrigerated food. Therefore, In the microbiological inspection of food hygiene, it must be paid attention to. The common detection of Listeria monocytogenes mainly relies on PCR technology. Although the sensitivity is high, it requires accurate temperature amplification, expensiv...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569
CPCG01N33/56911
Inventor 陈俊华潘家峰周丹华陈曼佳
Owner GUANGDONG INST OF ECO ENVIRONMENT & SOIL SCI