Application of real-time PCR for simultaneous identification and quantification of larval abalone

Abstract

A paucity of direct studies of marine invertebrate larval dispersal motivated the development of a high-throughput method for identification and quantification of pinto abalone ( Haliotis kamtschatkana) larvae in seawater. DNA extracted from sample retentate provided template to screen for species-specific cytochrome oxidase I (COI) mitochondrial DNA sequence via quantitative PCR (QPCR) technology. Primers and a dual-labeled probe were designed and used to identify and quantify DNA from the target species in blind tests of unknown samples alongside a standard template quantity series. Quantity estimates derived from QPCR standard curves were verified via direct enumeration of larvae using light microscopy. Multiplex reactions containing an internal positive control minimized underestimation of quantity and false negatives via partial or full PCR inhibition, respectively. Planned controlled field release and collection experiments to examine larval dispersion patterns via sampling over short and long postrelease times anticipate similar QPCR assays for other marine invertebrate species to aid investigations of larval dispersal in the marine environment.