A multiplex PCR-based protocol for identification and quantification of Vibrio harveyi-related species

Vibrios of the Harveyi clade are responsible for severe economic losses in the aquaculture industry worldwide, but so far no rapid method has been available to discriminate between the closely related species Vibrio campbellii, Vibrio harveyi, Vibrio owensii and Vibrio rotiferianus. Here, we describe a multiplex PCR assay capable of fast and reliable detection and identification of these species by amplification of three conserved protein-coding genes (topA, ftsZ and mreB). The developed species-specific primer sets produce PCR amplicons of unequal length and can be used separately or in combination. When combined with a 3-tube most-probable-number (MPN) based protocol, the multiplex PCR allows highly sensitive detection and enumeration in complex, aquaculture-relevant samples such as water, Artemia and spiny lobster (Panulirus ornatus) larvae. The described multiplex PCR represents a significant advance for aquaculture by providing an easy, fast and low-cost method for identification of several closely related pathogens that are difficult to discriminate by traditional approaches.