一种用于靶向金黄色葡萄球菌、无乳链球菌和大肠杆菌的序列切换细胞-SELEX DNA适配体。

A sequential toggle cell-SELEX DNA aptamer for targeting Staphylococcus aureus, Streptococcus agalactiae, and Escherichia coli bacteria.

作者信息

Kusumawati Arizah, Mustopa Apon Zaenal, Wibawan I Wayan Teguh, Setiyono Agus, Sudarwanto Mirnawati Bachrum

机构信息

Study Program of Veterinary Public Health, IPB Graduate School, IPB University, Bogor, Indonesia.

Research Center for Biotechnology, National Research and Innovation Agency (BRIN), Cibinong, Bogor, 16911, Indonesia.

出版信息

J Genet Eng Biotechnol. 2022 Jul 1;20(1):95. doi: 10.1186/s43141-022-00374-9.

DOI:10.1186/s43141-022-00374-9

PMID:35776386

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9249959/

Abstract

BACKGROUND

Mastitis is an inflammation of the mammary glands caused by a microbial infection. The common bacteria causing this infection in dairy farms are Staphylococcus aureus, Streptococcus agalactiae, and Escherichia coli. The aptamer is a new biosensor platform for detecting pathogens; however, its use for simultaneous detection of S. aureus, S. agalactiae, and E. coli bacteria has not been reported. This study's objective is to isolate and characterize polyclonal DNA aptamer with broad reactivity to the mastitis bacteria S. aureus, S. agalactiae, and E. coli using a sequential toggle cell-SELEX.

METHODS AND RESULTS

The DNA aptamer pool from SELEX 15 was inserted into the pGEM-T easy plasmid. Furthermore, the transformant clones were selected by PCR colony, plasmid isolation, and sequencing. Six DNA aptamers, consisting of S15K3, S15K4, S15K6, S15K13, S15K15, and S15K20 with a constant region and the right size of 81 bp were derived from the sequencing analysis. The secondary structure of the DNA was predicted using Mfold software. The DNA was analyzed with binding characteristics, including binding capacity and affinity (Kd), using qPCR. The results indicated aptamer S15K15 has the highest binding ability into S. agalactiae, while S15K13 performed binding capacity most to E. coli EPEC 4, and S15K3 has the highest capacity of binding to S. aureus BPA-12.

CONCLUSION

Aptamer S15K3 has the best binding characteristics on all three bacterial targets.

摘要

背景

乳腺炎是由微生物感染引起的乳腺炎症。奶牛场中引起这种感染的常见细菌有金黄色葡萄球菌、无乳链球菌和大肠杆菌。适配体是一种用于检测病原体的新型生物传感器平台；然而，尚未有关于其用于同时检测金黄色葡萄球菌、无乳链球菌和大肠杆菌的报道。本研究的目的是使用顺序切换细胞-SELEX技术分离并鉴定对乳腺炎细菌金黄色葡萄球菌、无乳链球菌和大肠杆菌具有广泛反应性的多克隆DNA适配体。

方法与结果

将SELEX 15的DNA适配体库插入pGEM-T easy质粒中。此外，通过PCR菌落、质粒分离和测序筛选转化体克隆。通过测序分析得到了六个DNA适配体，分别为S15K3、S15K4、S15K6、S15K13、S15K15和S15K20，它们具有恒定区域且大小合适，为81 bp。使用Mfold软件预测DNA的二级结构。使用qPCR分析DNA的结合特性，包括结合能力和亲和力（Kd）。结果表明，适配体S15K15对无乳链球菌的结合能力最强，而S15K13对大肠杆菌EPEC 4的结合能力最强，S15K3对金黄色葡萄球菌BPA-12的结合能力最强。