Characterization of Functional furVs as a Biomarker for Detection of Aquatic Pathogen Vibrio splendidus

Abstract

AbstractVibrio splendidus is an important opportunistic pathogen that can infect a broad range of aquatic animals including Apostichopus japonicus, leading to skin ulceration syndrome. In this study, fur gene of V. splendidus (furVs), the gene encoding ferric uptake regulator (Fur) of V. splendidus, was cloned and characterized. furVs possessed the signature ammonia acids of Vibrio sp., and its mRNA level was regulated by iron and coelomic fluid of A. japonicus. While the amino acid sequence of the furVs showed high homology with the compared Fur proteins of other Vibrio sp., the nucleotide sequence of furVs differed greatly from that of the other fur genes from Vibrio sp., based on which a pair of specific primers VSFurRTF3 and VSFurRTR3, was designed. A distinct DNA band with a length of 223 bp can be amplified from V. splendidus with great specificity. The detection limits for V. splendidus in seawater, homogenized tissues of epidermis, muscles, and coelomic fluid from A. japonicus, was 1 × 102, 1 × 105, 1 × 103, and 1 × 102 CFU/mL, respectively. Moreover, the detection limit was as low as 8 × 10−3 pg/μL when using DNA of V. splendidus as a template. Our results provide a simple and rapid polymerase chain reaction method for the sensitive and specific detection of V. splendidus.