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Annular mediated isothermal amplification-nano cadmium sulphide mark electrochemistry checking method for Listeria monocytogenes

A Listeria monocytogenes, ring-mediated isothermal technology, applied in biochemical equipment and methods, measuring devices, scientific instruments, etc., can solve the problems of cumbersome operation, time-consuming, and inability to meet the needs of disease prevention and control

Inactive Publication Date: 2008-09-10
QINGDAO UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There are three main methods for detecting Listeria monocytogenes in the past: 1. Physiological and biochemical identification technology, which is time-consuming and cumbersome to operate, and cannot meet the needs of disease control; 2. Enzyme-linked immunosorbent assay; 3. Polymerase chain reaction method (PCR method), which is faster and more sensitive than the previous two methods, but requires an expensive PCR instrument
There is no method for the detection of Listeria monocytogenes by combining the loop-mediated isothermal amplification method with the nano-label electrochemical DNA sensing technology

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Make nano cadmium sulfide (CdS)-probe sequence labeling solution according to the following formula:

[0041] (1) Labeling of the probe sequence by nano-CdS:

[0042] Add 3.84 μL of thioglycolic acid to 50 mL of CdCl at a concentration of 1.0 mmol / L 2 Mix well in the solution, adjust the pH value of the mixed solution to about 11 with 0.5mol / L NaOH solution, pass nitrogen gas to remove oxygen for 30min, slowly add 1.34mmol / L NaOH dropwise to the above mixed solution under magnetic stirring and nitrogen protection 2 S solution 30mL. After the dropwise addition, the stirring was continued for 24h, and the mixture gradually turned yellow. The CdS quantum dots prepared by the method have good stability, and no precipitation occurs after standing for three months under the condition of avoiding light.

[0043] Take 5.0mL CdS nano-sol for centrifugation and purification, wash with water and disperse in 2.0mL water, add 100μL 50.0mmol / L EDC, 100μL 50.0mmol / L NHS and 0.1mmol...

Embodiment 2

[0059] Use the following methods to detect the presence of Listeria monocytogenes in meat products:

[0060] (1) Preparation of nano-cadmium sulfide CdS-probe sequence labeling solution

[0061] Add 3.84 μL of thioglycolic acid to 50 mL of CdCl at a concentration of 1.0 mmol / L 2 Mix well in the solution, use 0.5mol / L NaOH solution to adjust the pH value of the mixed solution to about 11, pass nitrogen gas to remove oxygen for 30 minutes, slowly add 1.34mmol / L NaOH dropwise to the above mixed solution under magnetic stirring 2 S solution 30mL (nitrogen protection). After the dropwise addition, the stirring was continued for 24h, and the mixture gradually turned yellow. The CdS quantum dots prepared by the method have good stability, and no precipitation occurs after standing for three months under the condition of avoiding light.

[0062]Take 5.0mL CdS nano-sol for centrifugation and purification, wash with water and disperse in 2.0mL water, add 100μL 50.0mmol / L EDC, 100μL 5...

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Abstract

The invention relates to a method for detection Listeria monocytogenes with loop-mediated isothermal amplification-CdS nanoparticles labeling electrochemistry. The method is characterized in that the method comprises the following steps of: obtaining double-stranded target sequence of amplification reagent-Listeria monocytogenes to be detected by means of loop-mediated isothermal amplification reaction; pyrolyzing a double-stranded target sequence in water bath into a single-stranded target sequence, then self assembling and fixing at the surface of gold electrode, so that the target sequence modified electrode of the Listeria monocytogenes to be detected is obtained; afterwards, labeling the probe sequence coming from the Listeria monocytogenes by CdS nanoparticles to obtain a labeled probe sequence; carrying out molecular hybridization on the labeled probe sequence and the target sequence of the target sequence modified electrode, and carrying out electrochemical detection by simultaneity plating Hg anodic stripping method; detecting out cadmium ions corresponding to the species-specific genes (actA) of the detected Listeria monocytogenes. The method has the advantages of speediness, strong specificity, high sensitivity and convenient use.

Description

technical field [0001] The invention relates to a rapid gene detection method, specifically a method for detecting the genus-specific gene (actA) of Listeria monocytogenes by combining loop-mediated isothermal amplification (LAMP) technology and nano-label electrochemical DNA sensing technology ——Ring-mediated isothermal amplification-a method for the electrochemical detection of Listeria monocytogenes with nano-cadmium sulfide labeling. Background technique [0002] Listeria monocytohenes is a pathogen widely distributed in nature. It exists in soil, surface water, sewage, waste water, plants, silage, and rotten vegetables, so it is easy for animals to ingest it. The bacteria are transmitted through the oral-fecal route. According to reports, the carrying rate of Listeria monocytogenes in the feces of healthy people is 0.6-16%, 70% of people can carry bacteria for a short time, 4-8% of aquatic products, 5-10% of milk and its products, 30% More than 15% of meat products an...

Claims

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Application Information

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IPC IPC(8): C12Q1/04C12Q1/68G01N27/327
Inventor 孙伟高宏伟钟江华覃鹏焦奎
Owner QINGDAO UNIV OF SCI & TECH
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