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

A detection method, the technology of Enterobacter sakazakii, is applied in the fields of molecular biology and immunology, which can solve the problems of increasing the complexity of the detection process and losing the convenience of the test strip method, achieving high specificity, simple operation and accurate results Effect

Inactive Publication Date: 2014-04-16
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 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

Method used

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

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] 1 Materials and methods

[0055] Enterobacter sakazakii DNA

[0056] 1.2 Primer design

[0057] 1.3 PCR amplification system:

[0058]

[0059] Reaction conditions:

[0060]

[0061] 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.

[0062] 1.4 PCR specificity experiment

[0063] Using the established PCR reaction system for 1. Enterobacter sakazakii, 2. Yersinia enterocolitica, 3. Escherichia coli, 4. Salmonella, 5. Enterohaemorrhagic Escherichia coli O157, 6. Grape aureus coccus, 7. Listeria monocytogenes, 8. Shigella flexneri, 9 blank control (water), to verify its specificity.

[0064] 2 results

[0065] 2.1 PCR reaction system and conditions

[0066] HS Taq DNA polymerase from TAKARA Company was used, and the total reaction system was 20 μl. Detection was performed with a ...

Embodiment 2

[0070] 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.

[0071] 1 Materials and methods

[0072] Enterobacter sakazakii DNA

[0073] 1.2 Primer design

[0074] 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

[0075] 95 ℃ 5 minutes 94 ℃ 30 seconds 56.5 ℃ 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 3μl for nucleic acid test strip detection, take 3μl of the sample point on the sample...

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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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/10
CPCC12Q1/10C12Q1/6804C12Q2563/131C12Q2563/137C12Q2531/113Y02A50/30
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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