Preparation method and application of nucleic acid lateral flow test strip for detecting cronobacter sakazakii

Abstract

The invention discloses a rapid nucleic acid test strip detection kit for a food-borne pathogenic microorganism cronobacter sakazakii and an application method of the kit, and belongs to the fields of molecular biology and immunology. According to the kit and the application method thereof, a high-sensitivity and high-specificity polymerase chain reaction method for nucleic acid detection is combined with an immune colloidal gold rapid-detection technology for immunological detection, unique primers are designed and labeled, extracted target DNA (deoxyribonucleic acid) is subjected to specific amplification, and an amplification product is bound with a gold labeled antibody fixed on a test strip in a developing solution to form a stable and visible detection strip and a stable and visible quality control strip, so that main food-borne pathogenic microorganisms are rapidly and accurately detected.

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 Enterobacter sakazakii 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 Enterobacter sakazakii, Fu Shigella, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157: H7 and other pathogen...

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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 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 pseudoisocellular nucleobase derivatives and their application as primers or probes" provides a method using specially modified nucleotides as 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 method for adding internal control nucleic acid to pathogenic nucleic acid. "Semi-quantitative detection method" uses internal control probes to reduce interference. Among the above methods, the use of hot start technology and circular primers is a common method. The introduction of the needle into 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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