Direct multiplex PCR-NALFIA to inform marine conservation: Use of an innovative diagnostic tool for the detection of Ostrea edulis larvae

Abstract

The European oyster Ostrea edulis played a key role in the North Sea by providing several ecosystem functions and services. Today, O. edulis is classified as severely degraded or functionally extinct in Europe. Marine conservation is focusing on biogenic reef restoration, namely the restoration of O. edulis in Natura 2000 sites of the North Sea. The identification of oyster larvae related to natural spatfalls of restored reefs and monitoring of larval drift is a key aspect of marine protected area management. Morphological identification and distinction from other abundant bivalve larvae using microscopy is difficult. Existing molecular biological methods are expensive and bound to stationary laboratory equipment, or are inadequate in the visualization. In this study, we identified nucleic acid lateral flow immunoassay (NALFIA), a well-established tool in human pathogen diagnostics, as an efficient approach for point-of-care (POC) testing in marine monitoring. Based on the genetic sequence of the mitochondrial cytochrome b of O. edulis, forward and reverse primers were developed. The reverse primer was labelled with fluorescent dye FITC, forward primer with biotin. Reaction on the lateral flow stripe could be realized with a single O. edulis larva in direct PCR with multiplex primers in a portable PCR-cycler. The established NALFIA system can distinguish O. edulis larvae from Crassostrea gigas and Mytilus edulis larvae, respectively. This method offers new approaches in POC testing in marine research and monitoring. It gives quick and clear results, is inexpensive, and could be easily adapted to other species of interest.