Preparation and application of nucleic acid sidewise-flow test strip detection kit for detecting Bibrio parahemolyticus

Abstract

The invention discloses a rapid nucleic acid test strip detection kit of food-borne pathogenic microorganism Bibrio parahemolyticus and an application method of the kit, belonging to the technical field of bacteria classification inspection. According to the invention, a high-sensitivity high-specificity method of PCR reaction in nucleic acid detection is combined with an immuno gold staining rapid detection technology in immunological detection, specific amplification is carried out on target DNA by using a labeled specific primer through design of a specific primer, the amplified primer is combined with a gold labeled antibody immobilized on the test strip in a developing liquid so as to form a stable and visible detection zone and a quality control zone, thereby realizing the rapid and accurate detection of the Bibrio parahemolyticus.

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 Vibrio parahaemolyticus 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 food-borne disease risk factors, microbial food poisoning ranks first among the food-borne diseases prevalent in my country, and among the food-borne pathogenic microorganisms, the most typical pathogenic bacteria are Vibrio parahaemolyticus, Enterobacter sakazakii, Staphylococcus aureus, Listeria monocytogenes and other pathogenic bacteria...

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Problems solved by technology

[0005] Invention patent "Nick endonuclease nucleic acid constant temperature amplification rapid detection kit of Vibrio parahaemolyticus" (public number: CN201110366145) provides a constant temperature amplification Adding a detection method combined with gel electrophoresis or in situ hybridization, this type of method does not require cycle temperature PCR, which can significantly improve the detection sensitivity, but the detection method has not been significantly improved, and the operation is still too 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 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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