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SsDNA aptamer capable of specifically recognizing Weissella viridescens as well as screening method and application of ssDNA aptamer

A Weissella aptamer technology, applied in the field of specific recognition of Weissella viridans ssDNA aptamers and its screening, to achieve the effects of easy modification and labeling, strong specificity, and high affinity

Active Publication Date: 2021-09-07
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are currently no reports of aptamers related to Weissella viridans

Method used

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  • SsDNA aptamer capable of specifically recognizing Weissella viridescens as well as screening method and application of ssDNA aptamer
  • SsDNA aptamer capable of specifically recognizing Weissella viridescens as well as screening method and application of ssDNA aptamer
  • SsDNA aptamer capable of specifically recognizing Weissella viridescens as well as screening method and application of ssDNA aptamer

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] 1. The random library and primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0038] Random library:

[0039] 5'-AGC AGC ACA GAG GTC AGA TG-N40-CCT ATG CGT GCT ACC GTG AA-3';

[0040] 5'Carboxyfluorescein labeled upstream primer: 5'-FAM-AGC AGC ACA GAG GTC AGA TG-3';

[0041] 5' phosphorylation downstream primer: 5'-P-TTC ACG GTA GCA CGC ATA GG-3'.

[0042] The random library and primers were prepared into 10uM stock solution with TE buffer and stored at -20°C for later use.

[0043] 2. Culture of strains

[0044] Inoculate the activated Weissella viridans into MRS broth medium, culture at 37°C, and transfer the bacteria in the exponential phase (OD600=0.6) to the centrifuge tube according to the growth curve of Weissella viridans , centrifuge at 5000r / min, 4°C for 5min, discard the supernatant bacterial culture, and use binding buffer BB (10mmol / L Tris-HCl, 5mmol / L KCl, 100mmol / L NaCl, 5 mmol / L MgCl2 , pH 7.4) wash the white bacterial sediment...

Embodiment 2

[0056] (1) Aptamer affinity analysis

[0057] The 8 ssDNA sequences were dissolved to a concentration of 10 μM with binding buffer BB, and stored at -20°C for future use. The affinity of 8 ssDNA sequences to Weissella viridans was analyzed by flow cytometry. The concentration gradients of the eight ssDNA strips were 10, 20, 40, 80, 160 and 320nmol / L, respectively. After denaturation at 95°C for 10 minutes, they were immediately cooled in an ice bath for 10 minutes. Subsequently, the ssDNA solution was added to the Weissella viridis pellet washed three times with binding buffer BB, and incubated at 25° C. for 1 h with slow shaking. Then the W. viridans suspension was washed with binding buffer BB, and finally the complex of W. viridans-ssDNA was resuspended in 500 μL of binding buffer BB for flow cytometric analysis. The percentage of fluorescence intensity of the complex characterizes the affinity, and the dissociation constant K of each ssDNA is calculated by using GraphPad...

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Abstract

The invention relates to an SsDNA aptamer capable of specifically recognizing Weissella viridescens as well as a screening method and application of the ssDNA aptamer. The screening method comprises the following steps: constructing a random library and a primer pair, taking the Weissella viridescens as a forward screening strain and taking lactobacillus plantarum, lactobacillus brevis, leuconostoc mesenteroides, pediococcus pentosaceus and staphylococcus aureus as inverse screening strains, excluding sequences with low specificity, excluding non-specifically bound sequences through bovine serum albumin and tRNA, and obtaining an ssDNA library through screening; and sequencing the ssDNA library obtained by screening, selecting a sequence with a stable structure and relatively low free energy, and then screening out a sequence with high affinity and high specificity to the Weissella viridescens, so as to obtain the ssDNA aptamer capable of specifically recognizing the Weissella viridescens. The ssDNA aptamer has a potential application prospect in the aspect of accurately, quickly and sensitively detecting the Weissella viridescens.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a ssDNA aptamer for specifically recognizing Weissella viridans and its screening method and application. Background technique [0002] Weissella viridescens is a common dominant polluting microorganism in low-temperature meat products processing, and it is also a typical spoilage lactic acid bacteria in ultra-high pressure low-temperature meat products. Viridans has the ability to resist many unfavorable external conditions. In addition to being able to withstand ultra-high pressure treatment, other unfavorable conditions such as organic acid treatment and heat cannot completely kill it. Some meat products, such as sausages, vacuum-packed meat, smoked meat, etc., will turn green when exposed to oxygen. Weissella viride has become an important spoilage bacterium that threatens the quality and safety of meat products, and has caused huge economic losses to the meat processing industr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/569G01N33/58
CPCC12N15/115G01N33/56911G01N33/582C12N2310/16C12N2330/31
Inventor 王周平马鹏飞郭华麟段诺孙羽菡
Owner JIANGNAN UNIV
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