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Screening and application of oligonucleotide aptamer for specific recognition of Salmonella Typhimurium

An oligonucleotide, Salmonella technology, used in the determination/inspection of microorganisms, resistance to vector-borne diseases, DNA preparation, etc., can solve the problems of low sensitivity, high cost, instability, etc. Short-cycle, easy-to-mark effects

Active Publication Date: 2013-04-10
JIANGNAN UNIV
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Problems solved by technology

[0003] Traditional microbial testing is often time-consuming and insensitive; although the newly developed PCR technology can shorten the testing time, its sensitivity is still not high; immunological methods have the advantages of strong specificity, high sensitivity, and easy observation, but preparation Antibodies take a long time, are expensive, and unstable

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  • Screening and application of oligonucleotide aptamer for specific recognition of Salmonella Typhimurium
  • Screening and application of oligonucleotide aptamer for specific recognition of Salmonella Typhimurium
  • Screening and application of oligonucleotide aptamer for specific recognition of Salmonella Typhimurium

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Embodiment Construction

[0012] The following is a method for screening oligonucleotide aptamers specifically binding to Salmonella typhimurium by SELEX technology and an application for rapid detection of Salmonella typhimurium.

[0013] 1. Synthesize random single-stranded DNA library and primers (synthesized by IDT)

[0014] Random ssDNA library:

[0015] 5'-ATAGGAGTCACGACGACCAGAA-N40-TATGTGCGTCTACCTCTTGACTAAT-3'

[0016] A random ssDNA library with a length of 87 nt was constructed, with fixed primer sequences at both ends and a random sequence of 40 bases in the middle, with a library capacity of 10 14 Above; Primer I: 5'-ATAGGAGTCACGACGACCAGAA-3'; Primer II: 5'-ATTAGTCAAGAGGTAGACGCACATA-3', the random ssDNA library and primers were prepared into 100 μmol / L stock solution with TE buffer and stored at -20°C for later use.

[0017] 2. Cultivate Salmonella typhimurium in BBL liquid medium, and cultivate it on a shaker at 37°C until the logarithmic growth phase (OD 600 =0.3), collect the bacterial...

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Abstract

Belonging to the field of biotechnologies, the invention relates to screening and application of an oligonucleotide aptamer for specific recognition of Salmonella Typhimurium. Oligonucleotide aptamers able to specifically recognize Salmonella Typhimurium are obtained by a SELEX (systematic evolution of ligands by exponential enrichment) technique, and then a flow cytometer is employed to analyze the affinity and specificity of the aptamers, thus obtaining a Salmonella Typhimurium-recognizing oligonucleotide aptamer with the strongest affinity and the best specificity. The aptamer has wide application prospects in accurate, rapid and sensitive detection of Salmonella Typhimurium in food.

Description

technical field [0001] The present invention relates to screening and obtaining an oligonucleotide aptamer with high affinity and high specificity for Salmonella typhimurium by using SELEX technology (exponential enrichment ligand system evolution technology), and the oligonucleotide aptamer The application in identifying Salmonella belongs to the field of biotechnology. Background technique [0002] Salmonella Typhimurium (Salmonella Typhimurium), belonging to the Enterobacteriaceae, Gram-negative enterobacteriaceae. Salmonellosis is one of the zoonotic diseases of great significance in public health. In my country's inland areas, food poisoning caused by Salmonella has repeatedly ranked first. According to statistics, in my country's bacterial food poisoning, 70% - 80% are caused by Salmonella. Among the food poisoning around the world, Salmonella food poisoning in Britain and China ranks first. At present, worldwide, especially in developing countries, the number of pe...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C12N15/10C12Q1/68C12Q1/04
CPCY02A50/30
Inventor 王周平段诺吴世嘉
Owner JIANGNAN UNIV
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