Preparation and applications of nucleic acid lateral flow test strip kit for detecting EHEC O157

Abstract

The invention discloses preparation and applications of a nucleic acid lateral flow test strip kit for detecting EHEC O157, and belongs to the technical field of bacterial examination. According to the preparation and applications, a high-sensitivity and high-specificity method for polymerase chain reaction in a nucleic acid test is combined with an immune colloidal gold quick testing technology in immunological detection, specific amplification is carried out on a target DNA by virtue of a unique marked primer pair, and an amplification product is combined with a gold labeled antibody fixed on a test paper strip so as to form a stable and visible test band and a stable and visible quality control band, and thus the quick and accurate testing on Escherichia coli O157:H7 can be realized.

Description

technical field [0001] The invention belongs to the field of molecular biology and immunology, and relates to the preparation and application method of a lateral flow immune colloidal gold test strip kit of the nucleic acid amplification product of enterohaemorrhagic Escherichia coli (O-157) 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 risk factors of foodborne diseases, microbial food poisoning ranks first among the popular foodborne diseases in my country, and among the foodborne pathogenic microorganisms, the most typical pathogenic bacteria are Escherichia coli O157:H7, Enterobacter sakazakii, Staphylococcus aureus, Listeria monocytogenes and ot...

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Problems solved by technology

The invention patent "Loop-mediated isothermal amplification detection primer set, detection method and kit for Escherichia coli O157" (publication number: CN102943113) describes a rapid detection method using the loop-mediated isothermal amplification method combined with fluorescence color development. method, but the detection method has not been significantly improved, and the operation is more complicated

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 of 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 invention patent - "use 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 nucleobase derivatives and their application as primers or probes" provides a method using specially modified nucleotides as substrates In order to reduce 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 publication number is the invention patent of CN 101957373 A-"a semi-quantitative method of adding internal control nucleic acid to pathogenic nucleic acid "Detection 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 second 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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