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Aptamer for binding bifidobacterium breve as well as screening method and application of aptamer

A technology of Bifidobacterium breve and aptamer is applied in biochemical equipment and methods, microbial determination/inspection, DNA preparation, etc. The method is simple, the preparation cycle is short, and the operation is convenient.

Active Publication Date: 2016-08-10
苏州微克生活科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional detection method requires the cultivation of Bifidobacterium breve, which takes a long time, and the culture conditions have different effects on the morphology of Bifidobacterium breve, which leads to unreliable detection results and cannot meet the needs of rapid detection.
Molecular biology technology based on PCR technology improves the detection sensitivity and saves detection time to a certain extent, but this detection technology requires complex equipment and high technical requirements for detection personnel, so it is difficult to meet the needs of rapid detection

Method used

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  • Aptamer for binding bifidobacterium breve as well as screening method and application of aptamer
  • Aptamer for binding bifidobacterium breve as well as screening method and application of aptamer
  • Aptamer for binding bifidobacterium breve as well as screening method and application of aptamer

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] (1) The initial random single-stranded DNA (ssDNA) library and primer library sequences were chemically synthesized in vitro as follows:

[0040] 5'-AGCAGCACAGAGGTCAGATG-N40-CCTATGCGTGCTACCGTGAA-3' (Integrated DNA Technologies, IDT), constructed a random ssDNA library with a length of 80 nt, with fixed primer sequences at both ends and a random sequence of 40 nt in the middle, with a library capacity of 10 14 above.

[0041]Upstream primer: 5'-AGCAGCACAGAGGTCAGATG-3'

[0042] Downstream primer: 5'-TTCACGGTAGCACGCATAGG-3'

[0043] 5' phosphorylation downstream primer: 5'-P-TTCACGGTAGCACGCATAGG-3'

[0044] The random ssDNA library and primers were prepared into 100uM stock solution with TE buffer and stored at -20°C for future use.

[0045] (2) SELEX screening

[0046] The Bifidobacterium breve that is cultivated to the logarithmic phase is used binding buffer (50mmol / L Tris-HCl (pH 7.4), 5mmol / L KCl, 100mmol / L NaCl, 1mmol / L MgCl 2 ) was washed twice to remove the cu...

Embodiment 2

[0048] The interaction between single-stranded nucleotide bases can make it form a certain spatial structure, and these structures are easy to realize conformational binding with various target molecules. Based on this conformational recognition feature, a sufficiently large capacity (10 14 The nucleic acid library of the above) can contain as many three-dimensional structures as possible, so it is possible to screen aptamers with high affinity and high specificity for any type of target.

[0049] Based on the above principles, the inventors in this example propose a set of nucleic acid aptamer sequences that specifically bind to Bifidobacterium breve through screening through improved whole-Bacterium SELEX technology. The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0050] The nucleic acid aptamer screening method for the specific binding of Bifidobacterium breve is roughly as follows:

[0051] (...

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Abstract

The invention provides an aptamer for binding bifidobacterium breve as well as a screening method and an application of the aptamer. The aptamer comprises a nucleotide sequence which is higher than 65% in bifidobacterium breve binding rate with a sequence shown as 5'-AGCAGCACAGAGGTCAGATG-N40-CCTATGCGTGCTACCGTGAA-3', wherein N represents any one of base groups A, T, C and G, and N40 stands for a base group that a random fragment length includes 40 base groups. The high-quality aptamer for the bifidobacterium breve disclosed by the invention is relatively high in binding capacity with the bifidobacterium breve and is good in specificity; and the aptamer can be widely applied to various fields of biotechnology related to the bifidobacterium breve.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to an aptamer for bifidobacterium breve, a screening method and a method for utilizing the aptamer. Background technique [0002] For a long time, Bifidobacterium and its probiotics, as probiotics, have attracted more and more attention from academic circles and businessmen due to their unique properties. Bifidobacteria can not only inhibit the growth of intestinal pathogenic bacteria, thereby regulating and improving the intestinal flora, but also can improve lactose intolerance, prevent intestinal infection, lower serum cholesterol, promote immunity and exert antioxidant properties, etc. effect. Yogurt containing bifidobacteria has been popular in Europe and Japan for many years, and sales are still growing. Our country has also developed a variety of bifidobacteria products, such as yogurt, milk powder, beverages and other products containing bifidobacteria, and the sale...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C12N15/10
CPCC12N15/1048C12N15/115C12N2310/16C12Q2531/113C12Q2527/125
Inventor 陈卫张灏胡陆军赵建新田丰伟王刚张秋香范大明翟齐啸赵国忠
Owner 苏州微克生活科技有限公司
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