Preparation method and application of nucleic acid lateral flow test strip for detecting listeria monocytogenes

Abstract

The invention discloses a rapid nucleic acid test strip detection kit for foodborne pathogenic microorganism-listeria monocytogenes and an application method thereof, belonging to the technical field of bacterial examination. The stable and visible detection zone and quality control zone are formed by combining the high-sensitivity and high-specificity polymerase chain reaction method in nucleic acid detection with the immune colloidal gold rapid detection technology in immunological detection, designing unique primers, labelling the primers, specifically amplifying the extracted target DNA and combining the amplification product with gold labelled antibodies immobilized on the test strip in a developing solution, thus achieving rapid and accurate detection of the main foodborne pathogenic microorganism.

Description

technical field [0001] The invention belongs to the fields of molecular biology and immunology, and relates to a preparation and application method of a lateral flow immune colloidal gold test strip kit of a nucleic acid amplification product of Listeria monocytogenes in food and processing raw materials. Background technique [0002] In order to effectively control foodborne diseases caused by pathogenic microorganisms in food production and import and export trade, and ensure public safety in the food field, it is necessary to develop sensitive, convenient and accurate detection methods for foodborne pathogenic microorganisms. Among the foodborne disease risk factors, microbial food poisoning ranks first among the foodborne diseases prevalent in my country, and among the foodborne pathogenic microorganisms, the most typical pathogenic bacteria are Listeria monocytogenes, Shigella flexneri, Staphylococcus aureus, Escherichia coli O157: H7 and other pathogenic bacteria. List...

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Problems solved by technology

In terms of primer optimization, the invention patent with the publication number CN 102146432 A - "A method for reducing the dimer of a pair of partially homogeneous primers" describes a primer design method with a short palindromic sequence at the 5' end of the primer, The primer self-cyclizes at room temperature to avoid the formation of heterodimers. The invention patent with the publication number CN 102719547 A - "Reagent for Real-time Fluorescent Quantitative PCR for Detecting HER2 Gene Expression Level" also uses a similar method for real-time quantitative PCR Amplification; Publication No. CN 101842494 A patent for invention - "using chimeric primers to reduce heterodimer formation" describes a method of using chimeric primers to amplify; in the optimization of the reaction substrate, The invention patent of publication number CN 101171343A "3' modified oligonucleotides containing pseudo-isocytosine base derivatives and their application as primers or probes" provides a method of using specially modified nucleotides as substrates to The method for reducing the formation of primer dimers, the use of probes or the introduction of internal control probes can also reduce the interference of non-specific amplification, the invention patent of which the publication number is CN 101957373 A-"a semi-quantitative detection of pathogenic nucleic acid by adding internal control nucleic acid The method "is to use internal control probes to reduce interference. Among the above methods, the use of hot start technology and circular primers is a common method, and the rest of the methods either use specially synthesized substrates and substrates, or need to introduce the first The secondary hybridization process will increase the complexity of the detection process, especially the probe hybridization method, which loses the convenience of the test strip method due to the need for an incubation process

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