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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: 2013-07-31
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 the conventional method, use the TIANamp Bacteria DNA Kit kit to extract the DNA in the milk sample;

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

[0061] (3) Add 2 nmol of a designed highly specific 5'-end amino-modified primer 1 (see Table 1 for the sequence) and 100 μL of ligation reaction solution (0.1M 2-? Methyrethanesulfonic acid (MES), 0.25% Tween-20, 10 mM carbodiimide hydrochloride (EDC)) were co-incubated for 5 hours at room temperature with a shaker gently shaking.

[0062] (4) After the reaction was completed, the prepared magnetic primers were separated using a magnetic separation pen, and washed twice with washing buffer (1 times standard 10 mM, PBS pH 7.4, 0.25% Tween-20). Finally, the synthesized magnetic primers were placed in 100 μL stor...

Embodiment 2

[0072] Example two Listeria monocytogenes detection sensitivity experiment

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

Embodiment 3

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

[0075]Get respectively 100ng Listeria monocytogenes (Listeria monocytogenes, CMCC54007) genome, 100ng Escherichia coli (E.coli O157:H7, GW1.2020) genome, 100ng Salmonella (Salmonella enterica, CMCC50040) genome, according to the method described in Example 1 The measured electrochemical signal values ​​are as Figure 5 shown by Figure 5 It can be seen that when there are Escherichia coli, Salmonella and Listeria monocytogenes genomes of the same mass concentration in the sample, only when the target genome exists, can a higher electrochemiluminescence signal be obtained, thus it can be judged that the method of the present invention can detect 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, in particular to a method for detecting food pathogenic bacteria by an electrochemiluminescence gene sensor based on magnetic in-situ amplification. Background technique [0002] As a serious public health problem, foodborne pathogens are one of the main reasons affecting food safety. It seriously endangers people's life and health and causes great economic losses. According to the information released by the World Health Organization in March 2002, billions of cases of food-borne diseases occur every year in the world, and about 1.7 million children die each year from diarrhea caused by food-borne pathogenic bacteria contamination. About 20,000 to 40,000 cases of foodborne diseases are reported in my country every year, but according to experts' estimates, this number is less than 10% of the actual number. The data from the Guangdong Provincial Department of Hea...

Claims

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Application Information

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