Method for detecting food pathogen by electrochemical luminescence gene sensor on basis of magnetic in-situ amplification

A technology of luminescent genes and pathogenic bacteria, applied in the field of electrochemiluminescence gene detection

Active Publication Date: 2011-10-19
SOUTH CHINA NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This technology designs three PCR functional primers for the target gene

Method used

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  • Method for detecting food pathogen by electrochemical luminescence gene sensor on basis of magnetic in-situ amplification
  • Method for detecting food pathogen by electrochemical luminescence gene sensor on basis of magnetic in-situ amplification
  • Method for detecting food pathogen by electrochemical luminescence gene sensor on basis of magnetic in-situ amplification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] (1) According to conventional methods, use TIANamp Bacteria DNA Kit to extract DNA from milk samples;

[0060] (2) Take 1 mg of magnetic particles with a particle size of 100 nm and surface modified with carboxyl groups, and wash them with 200 μL of 0.01M sodium hydroxide solution at room temperature for 2 times, and then rinse with deionized water once.

[0061] (3) To the washed magnetic particles, add a highly specific 5'end modified primer 1 with an amino group (see Table 1 for the sequence) 2nmol and 100μL pH5.0 ligation reaction solution (0.1M 2-? Incubate for 5 hours at room temperature with phytoethanesulfonic acid (MES), 0.25% Tween-20, 10mM carbodiimide hydrochloride (EDC), and shake the mixer slightly.

[0062] (4) After the reaction is completed, use a magnetic separator to separate the prepared magnetic primers, and wash them twice with a washing buffer (1 times standard 10 mM, pH 7.4 PBS, 0.25% Tween-20). Finally, the synthesized magnetic primers were placed in ...

Embodiment 2

[0072] Example 2 Detection sensitivity test of Listeria monocytogenes

[0073] Take 10 respectively 5 pg, 10 4 pg, 10 3 pg, 10 2 pg, 50pg, 10pg Listeria monocytogenes (CMCC54007) genome, the electrochemical signal value measured according to the method described in Example 1 is as follows Figure 4 Shown by Figure 4 It can be seen that when the concentration of the target genome fragment of Listeria monocytogenes is equal to 0.5pg / ul, the electrochemiluminescence signal is still greater than the threshold 299cps (the luminescence average of the three groups of negative samples is 230cps, and the 3-fold standard deviation between samples is 66cps), so it can It is judged that the detection target gene of the present invention has very good sensitivity.

Embodiment 3

[0074] Example 3 Electrochemiluminescence verification experiment for the specificity of Listeria monocytogenes detection

[0075] Take 100ng Listeria monocytogenes (CMCC54007) genome, 100ng Escherichia coli (E.coli O157:H7, GW1.2020) genome, and 100ng Salmonella enterica (CMCC50040) genome respectively, according to the method described in Example 1. The measured electrochemical signal value is as Figure 5 Shown by Figure 5 It can be seen that when the genomes of Escherichia coli, Salmonella and Listeria monocytogenes at the same mass concentration are respectively present in the sample, only when the target genome is present can a higher electrochemiluminescence signal be obtained, which can be judged by the method of the present invention. Listeria monocytogenes has very good specificity.

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Abstract

The invention discloses a method for detecting food pathogen by an electrochemical luminescence gene sensor on the basis of magnetic in-situ amplification. The method comprises the following steps: designing three PCR (Polymerase Chain Reaction) primers by using highly conserved DNA (deoxyribonucleic acid) sequence of food pathogen as a detection target segment; preparing a magnetic primer from the primer 1, preparing a TBR (tris (bipyridine) ruthenium) primer from the primer 2, and using the primer 3 as a common PCR primer, wherein when a target gene segment exists in the detection system, the magnetic primer can be amplified as the PCR process goes on, and the magnetic particles can directly have a TBR-modified double-chain nucleic acid product; when no target gene segment exists, the magnetic primer can not be amplified, and thus, no product with signal molecules can appear; and carrying out magnetic separation and in-situ electrochemical luminescence detection. The method can be used for quickly, sensitively, safely and specifically detecting the target gene segment, and is very suitable for popularization in the actual detection process.

Description

Technical field [0001] The invention belongs to the technical field of electrochemiluminescence gene detection, and particularly relates to a method for detecting food pathogenic bacteria by an electrochemiluminescence gene sensor based on magnetic in situ amplification. Background technique [0002] Food-borne pathogens as a serious public health problem are one of the main reasons affecting food safety. It seriously endangers people's lives and health and causes major economic losses. According to the information released by the World Health Organization in March 2002, there are billions of cases of foodborne diseases worldwide each year, and about 1.7 million children die of diarrhea caused by contamination by foodborne pathogens. my country reports about 2-4 million cases of food-borne diseases each year, but according to expert estimates, this number is still less than 10% of the actual number. Data from the Guangdong Provincial Department of Health in 2008 also showed tha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/01C12R1/42
Inventor 邢达朱啸
Owner SOUTH CHINA NORMAL UNIVERSITY
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