Isolation and Characterization of a ssDNA Aptamer against Major Soluble Antigen of Renibacterium salmoninarum

Abstract

Bacterial kidney disease (BKD) is a major health problem of salmonids, affecting both wild and cultured salmon. The disease is caused by Renibacterium salmoninarum (Rs), a fastidious, slow-growing and strongly Gram-positive diplobacillus that produces chronic, systemic infection characterized by granulomatous lesions in the kidney and other organs, often resulting in death. Fast detection of the pathogen is important to limit the spread of the disease, particularly in hatcheries or aquaculture facilities. Aptamers are increasingly replacing conventional antibodies as platforms for the development of rapid diagnostic tools. In this work, we describe the first instance of isolating and characterizing a ssDNA aptamer that binds with high affinity to p57 or major soluble antigen (MSA), the principal antigen found on the cell wall surface of Rs. Specifically, in this study a construct of the full-length protein containing a DNA binding domain (MSA-R2c) was utilized as target. Aptamers were isolated from a pool of random sequences using GO-SELEX (graphene oxide-systematic evolution of ligands by exponential enrichment) protocol. The selection generated multiple aptamers with conserved motifs in the random region. One aptamer with high frequency of occurrence in different clones was characterized and found to display a strong binding affinity to MSA-R2c with a Kd of 3.0 ± 0.6 nM. The aptamer could be potentially utilized for the future development of a sensor for rapid and onsite detection of Rs in water or in infected salmonids, replacing time-consuming and costly lab analyses.

Generation and Selection of Specific Aptamers Targeting Brucella Species through an Enhanced Cell-SELEX Methodology

“…The mixture was heated at 95 °C for 10 min to elute the bound aptamer sequences. The quantification of aptamers bound to the Brucella cells was carried out by qPCR analysis [ 52 , 96 , 97 , 98 ] (StepOne, Applied Biosystems) using SensiFAST SYBR ® mix (2X) kit (Bioline, Cat. No.…”