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

A test strip and feed technology, applied in the fields of molecular biology and immunology, can solve the problems of increasing the complexity of the detection process and losing the convenience of the test strip method, and achieve the effects of easy promotion, high specificity, and intuitive technology

Inactive Publication Date: 2014-04-30
ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
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  • Application Information

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...

Method used

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

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] 1 Materials and methods

[0052] Material: donkey mitochondrial DNA

[0053] 1.2 Primer design

[0054] 1.3 PCR amplification system:

[0055] 1.3 PCR amplification system:

[0056] Template DNA: donkey mitochondrial DNA 1.0 μl Primer F / R 0.8 μl dNTP 2.0 μl 10X PCR buffer 2.0 μl HS Taq DNA polymerase 0.2 μl ddH2O 13.2 μl total capacity 20 μl

[0057] Reaction conditions:

[0058] 95 ℃ 5 minutes 94 ℃ 30 seconds 55 ℃ 30 seconds 72 ℃ 30 seconds 72 ℃ 5 minutes 30 cycles in total

[0059] At the same time, take 5 μl for nucleic acid test strip detection, take 5 μL of the amplification product, add it to 95 μL of developing solution for detection and spot on the sample pad, and observe the results after 5 minutes.

[0060] 1.4 PCR specificity experiment

[0061] Using the established PCR reaction system for 1: mouse; 2: donkey; 3: rabbit; 4: horse; 5: sheep; 6: dog; ...

Embodiment 2

[0068] The sensitivity of the nucleic acid test strip detection method, the PCR template is 1 / 10, 1 / 10 2 , 1 / 10 3 , 1 / 104 , 1 / 10 5 , 1 / 10 6 , 1 / 10 7 , 1 / 10 8 , 1 / 10 9 , 1 / 10 10 After dilution, the PCR system was established for amplification.

[0069] 1 Materials and methods

[0070] Donkey mtDNA

[0071] 1.2 Primer design

[0072] 1.3 PCR amplification system:

[0073] Template DNA: plasmid DNA 10 μl Primer F / R 0.8 μl dNTP 2.0 μl 10X PCR buffer 2.0 μl HS Taq DNA polymerase 0.2 μl ddH2O 4.2 μl total capacity 20 μl

[0074] Reaction conditions:

[0075] 95 ℃ 5 minutes 94 ℃ 30 seconds 57 ℃ 30 seconds 72 ℃ 80 seconds 72 ℃ 5 minutes 30 cycles in total

[0076] Take 5μl respectively for agarose gel electrophoresis. The gel electrophoresis conditions are: 1X TBE buffer, voltage 100V, electrophoresis time 30 minutes. At the same time, take 5μl for nucleic acid test s...

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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...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6804C12Q2563/137C12Q2563/131C12Q2531/113
Inventor 郑文杰贺艳陈其勇程瑜张宏伟张灿奚文辉杜敬韩宇宁尹长城刘斯奇李宏虹张亚莲
Owner ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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