Preparation method of nucleic acid lateral flow test strip for detecting yersinia enterocolitica and application thereof

A technology for enterocolitis and Yersinia, applied in the fields of molecular biology and immunology, can solve the problems of losing the convenience of the test strip method and increasing the complexity of the detection process, achieving high specificity, simple operation, Technically Intuitive Effects

Inactive Publication Date: 2014-05-14
ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • 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 "3' modified oligonucleotides containing pseudoiso

Method used

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  • Preparation method of nucleic acid lateral flow test strip for detecting yersinia enterocolitica and application thereof
  • Preparation method of nucleic acid lateral flow test strip for detecting yersinia enterocolitica and application thereof
  • Preparation method of nucleic acid lateral flow test strip for detecting yersinia enterocolitica and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] 1 Materials and methods

[0051] Yersinia enterocolitica DNA

[0052] 1.2 Primer design

[0053] 1.3 PCR amplification system:

[0054]

[0055] Reaction conditions:

[0056]

[0057] At the same time, take 3 μl for nucleic acid test strip detection, take 3 μL of the amplification product and spot it on the sample pad, add it to 97 μL of developing solution for detection, and observe the result after 5 minutes.

[0058] 1.4 PCR specificity experiment

[0059] Use the established PCR reaction system for A. enterocolitica Yersinia, B. Salmonella, C. Enterobacter sakazakii, D. Escherichia coli, E.O-157, 2. Negative control bacteria, 1) Grape aureus Coccus 2) Listeria monocytogenes, 3) Shigella flexneri, 3. Negative control (water) to verify its specificity.

[0060] 2 results

[0061] 2.1 PCR reaction system and conditions

[0062] The hot-start Taq DNA polymerase from TAKARA Company was used, and the total reaction system was 20 μl. Detection was performed w...

Embodiment 2

[0066] The sensitivity of the nucleic acid test strip detection method, the PCR template is 1, 1 / 10, 1 / 10 2 , 1 / 10 3 , 1 / 10 4 , 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 to amplify.

[0067] 1 Materials and methods

[0068] Yersinia enterocolitica DNA

[0069] 1.2 Primer design

[0070] 1.3 PCR amplification system:

[0071]

[0072] Reaction conditions:

[0073]

[0074] 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 3μl for nucleic acid test strip detection, take 3μl of the sample point on the sample pad and add it to 97μL of developing solution for detection, and observe the results after 5 minutes.

[0075] 2 results

[0076] 2.1 PCR reaction system and conditions

[0077] HS Taq DNA polymerase from TAKARA Company was used, and the total reaction system was 20 μl...

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Abstract

The invention discloses a preparation method of a quick nucleic acid test strip detection kit for food-borne pathogenic microorganism-yersinia enterocolitica and an application thereof, and also discloses a design method of a primer for specific nucleic acid PCR (polymerase chain reaction) amplification for yersinia enterocolitica, and a method for detecting a PCR amplification product with the nucleic acid test strip for nucleic acid lateral flow and an application. The method disclosed by the invention can be used for detecting the amplification product of the target nucleotide sequence of pathogenic microorganisms from other sources by use of the specific nucleotide sequence amplification product, wherein the specificity is guaranteed by the amplification primer.

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 Yersinia enterocolitica 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 prevalent food-borne diseases in my country, and among the food-borne pathogenic microorganisms, the most typical pathogenic bacteria is enterocolitis Yersinia Bacteria, Shigella flexneri, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157: H7 ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/10
CPCC12Q1/10C12Q1/6804C12Q2531/113C12Q2563/131C12Q2565/625
Inventor 郑文杰李宗梦张宏伟刘培曲鹏奚文辉郝育杰尹长城刘斯奇
Owner ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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