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A set of oligonucleotide aptamers capable of specifically recognizing streptococcus agalactiae

A technology of Streptococcus lactis and oligonucleotides, applied in the biological field, can solve time-consuming and difficult problems, and achieve the effects of low preparation cost, simple operation, and stable single-stranded oligonucleotides

Active Publication Date: 2013-04-03
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In essence, most of the surface antigens contained in the same genus are the same, with only slight differences. It is obviously a time-consuming and arduous task to find these differences and prepare corresponding specific antibodies

Method used

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  • A set of oligonucleotide aptamers capable of specifically recognizing streptococcus agalactiae
  • A set of oligonucleotide aptamers capable of specifically recognizing streptococcus agalactiae
  • A set of oligonucleotide aptamers capable of specifically recognizing streptococcus agalactiae

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Embodiment 1

[0016] Example 1: Competition SELEX screening of Streptococcus agalactiae-specific binding oligonucleotide aptamers

[0017] 1. In vitro chemical synthesis of initial random single-stranded DNA (ssDNA) library and primers (completed by Integrated DNA Technologies, USA), the sequence is as follows: 5'-ATAGGAGTCACGACGACCAG(40N)TATGTGCGTCTACCTCTTGA-3'; a random ssDNA library with a length of 80nt was constructed, The two ends are fixed primer sequences, the middle is a random sequence of 40 bases, and the library capacity is 10 14 above;

[0018] Primer I: 5'-ATAGGAGTCACGACGACCAG-3';

[0019] Primer II: 5'-TCAAGAGGTAGACGCACATA-3';

[0020] 5'phosphorylation downstream primer: 5'-P-TCAAGAGGTAGACGCACATA-3';

[0021] The random ssDNA library and the two primers were made into a 100 μM stock solution in TE buffer and stored at -20°C for future use.

[0022] 2. Conditions for PCR amplification and Lambda exonuclease digestion to prepare single-strand sub-libraries

[0023] The sy...

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Abstract

The invention discloses a set of oligonucleotide aptamers capable of specifically recognizing streptococcus agalactiae, and belongs to the field of food hygiene and clinical medicine detection. Aiming at the defects that no oligonucleotide aptamers for the streptococcus agalactiae are in the prior art, the oligonucleotide aptamers provided by the invention combine an SELEX technology with the streptococcus agalactiae, a single stranded secondary library is prepared by incubating, cleaning, dissociating, amplifying, and digesting by using lambda exonuclease again and again for 12 rounds, the oligonucleotide aptamers capable of specifically combining with the streptococcus agalactiae are selected from a single stranded DNA random library; and three aptamers with the best effect are obtained by sequencing, affinity analysis and specificity analysis, wherein the nucleotide sequences are selected from 1 to 3 in the sequence table. The set of the oligonucleotide aptamers provide specific and efficient recognition ligands for analyzing and detecting the streptococcus agalactiae in the food hygiene and clinical blood samples and novel choices for developing and replacing a conventional method that detects the streptococcus agalactiae depending on antibodies.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the preparation of a nucleic acid aptamer with high specificity and high affinity for S. , providing a scientific basis and a theoretical basis for the application of the nucleic acid aptamer in the detection of Streptococcus agalactiae. Background technique [0002] Streptococcus agalactiae, also known as Group B of streptococcus (GBS), can not only cause mastitis in dairy cows, but also reduce the milk production and milk quality of dairy cows, causing huge economic losses to the dairy industry; At the same time, it is also the most important infectious pathogen that causes morbidity and death in human newborns and pregnant women. Boe reported that GBS infection causes more than 1,300 maternal deaths in the United States every year, and Heath et al. reported that the incidence of GBS infection in newborns in the United Kingdom was 0.72 / 1000 live births. Therefore, how to quickly ...

Claims

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Application Information

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IPC IPC(8): C12N15/115C12Q1/68C12Q1/14C12R1/46
Inventor 王周平王鑫段诺吴世嘉夏雨马小媛
Owner JIANGNAN UNIV
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