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.
Highlights
Renibacterium salmoninarum (Rs) is the causative agent for bacterial kidney disease (BKD) which affects wild and cultured salmonids and has a serious impact on the salmonid industry worldwide [1]
major soluble antigen (MSA) and its breakdown products are the principal constituents of the extracellular protein (ECP) fraction released by Rs into fish tissues and body fluids and the surrounding environment [17]
Because MSA is currently used as the antigen for detection of Rs by enzyme-linked immunosorbent assay (ELISA), it is considered a promising target for aptamer screening due to its uniqueness and high abundance
Summary
Renibacterium salmoninarum (Rs) is the causative agent for bacterial kidney disease (BKD) which affects wild and cultured salmonids and has a serious impact on the salmonid industry worldwide [1]. Traditional methods to detect Rs are based on bacterial culture of infected tissues. While this method can be very sensitive, it is time consuming due to the very slow growth of the bacterium. The putative sensitivity of ELISA has been estimated to be several hundred cells per reaction [4] or approximately 9 × 106 cells/g of tissue [5]. These methods focus primarily on tissues such as kidney or ovarian fluid, requiring that fish be sacrificed and are not suitable for onsite detection of the bacterium
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