Application of Environmental Sample Processor (ESP) methodology for quantifying Pseudo-nitzschia australis using ribosomal RNA-targeted probes in sandwich and fluorescent in situ hybridization formats

Dianne I. Greenfield, Roman Marin, Scott Jensen, Eugene Massion, Brent Roman, Jason Feldman, Christopher A. Scholin

Limnol. Oceanogr. Methods 4:426-435 (2006) | DOI: 10.4319/lom.2006.4.426

ABSTRACT: In this contribution, we asses the application of methodology used in a novel in situ remote sampling platform, the Environmental Sample Processor (ESP), for the identification and enumeration of a harmful algal species, the domoic-acid producing diatom Pseudo-nitzschia australis, using two molecular assays: a sandwich hybridization assay (SHA) and fluorescent in situ hybridization (FISH). Both the SHA and FISH assays were initially designed as laboratory-based methods that are now emulated in the ESP. Response of the SHA to a range of concentrations of P. australis using the laboratory (96-well plate) and ESP (DNA probe array) formats showed that the two were highly correlated. Enumeration of cells filtered and archived for FISH using a manifold designed for laboratory applications agreed well with counts of cells filtered and archived in the ESP at ≥ 2.5 x 104 cells L-1. At lower concentrations, it becomes statistically unlikely to derive a reliable abundance estimate suggesting that FISH is better suited for qualitative analyses unless the target organism is very abundant. We also assessed the suitability of an oligonucleotide as synthetic target sequence to act as a SHA reagent quality control and internal standard for the plate assay. This was successful, but the probe and/or associated reagents were stable for only ~60 d. Our results show that the ESP is capable of detecting P. australis in near real-time and also supports whole cell archival, making it a potentially useful tool for future research and monitoring initiatives.