Development of single and multispecies detection methods for the surveillance and monitoring of marine pests in New Zealand.

New Zealand's current surveillance programme for marine pests does not include methods for identification of organisms in the water column, therefore dispersive forms go undetected. Molecular methods provide an opportunity to detect dispersive forms thereby contributing to a more robust surveillance programme. Additionally, New Zealand has become a signatory to the IMO Ballast Water Convention and there is scope for developing enumeration methods for detection of viable organisms as well as species-specific molecular probes for indicator bacteria. We describe here the outcome of experiments testing the effects of various matrices (e.g. sediment, biofilms, benthic assemblage, seawater) against a quantitative PCR (QPCR) assay developed for Potamocorbula amurensis. The limit of detection of the assay for sediments and benthic assemblages ranged between one to five larvae per 10 g wet weight. We also report a QPCR assay for Vibrio cholerae, one of the indicator species in the Ballast Water Convention. Quantitative PCR of seawater spiked with different concentrations of culture showed that assay sensitivity was insufficient to detect the regulatory limit of 1 colony forming unit (CFU) per 100 mL. More recent work suggests that sufficient sensitivity can be achieved with cultivation based methods (most probable number-QPCR [MPN-QPCR]). We are currently appraising several multispecies detection methods including micro-arrays and next generation sequencing for their suitability in marine pest detection. The advantages and disadvantages of each are discussed herein. We highlight the need to consider issues relating to sampling design when using molecular methods for surveillance and compliance monitoring.