Lateral flow test paper strip detection kit for detection of donkey source component in food and feed, and application thereof

Abstract

The present invention discloses a preparation method of a rapid nucleic acid detection kit for a donkey (Equusafricanusasinus) component in food and feed, and an application method thereof, and belongs to the field of molecular biology and immunology. According to the invention, the high sensitivity and high specificity polymerase chain reaction method in nucleic acid detection and the immune colloidal gold rapid detection technology in immunology detection are combined, the unique primers are designed, the primers are labeled, specific amplification is performed on the extracted target DNA, and the amplified product is bound with gold-labeled antibody immobilized on test paper strip in a spreading solution so as to form the stable and visible detection zone and the quality control zone, such that the rapid and accurate detection on the donkey source component in food and feed is achieved.

Description

technical field [0001] The invention belongs to the fields of molecular biology and immunology, and relates to PCR nucleic acid amplification of donkey-sourced DNA in food and feed, and preparation and application methods of a lateral flow immune colloidal gold test strip kit. Background technique [0002] In order to prevent the spread of bovine spongiform encephalopathy (BSE) through beef and beef bones added to animal feed, the European Union and the United States in 1994 and 1997 respectively enacted a ban on adding ruminants to ruminant feed Regulations for protein of animal origin, the European Union in 2000 extended this ban to prohibit feeding processed mammalian, bird and fish protein ingredients to animals intended for food. The "Administrative Measures for the Safety and Hygiene of Animal-derived Feed Products" promulgated by the Ministry of Agriculture of my country in 2004 stipulates that the use of animal-derived feed products (except milk and dairy products) i...

AI Technical Summary

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 "Containing 3' modified oligonucleotides of pseudo-isocytosine nucleic acid base 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

Images