Application of a sandwich hybridisation assay for rapid detection of the Northern Pacific seastar, Asterias amurensis

Abstract

A sandwich hybridisation assay (SHA) has been developed to detect Asterias amurensis (Lu¨tken 1871), commonly known as the northern Pacific seastar, which is an unwanted, and as yet undetected, pest in New Zealand waters. Probes in SHA format have been developed that target the ribosomal RNA internal transcribed spacer region of A. amurensis. The successful assay utilises the capture probe ITS-C2-110 and the combined signal probes ITS-S1-93 and ITS-S3-240. It has been successfully tested for specificity, including cross-reactivity testing against the common and related seastars, Patiriella regularis (Verrill 1867), P. mortenseni (O'Loughlin et al. 2002), Coscinasterias muricata (Verrill 1867) and Stichaster australis (Verrill 1871). The SHA is a rapid and potentially portable RNA-based test, and opens up the opportunity for direct analysis of water samples, for example, portside during surveillance of marine pests.

Development of a sandwich hybridization assay for the identification and quantification of red drum (Sciaenops ocellatus) eggs: a novel tool for fishery research and management

“…KP739311 to KP739346). Consequently, capture probe development efforts focused on the more variable and noncoding ITS region, which has been previously used to develop capture probes for the starfish Asterias amurensis (Smith et al 2011). The ITS and adjacent 5S regions were amplified using the rDNA primers CS249 and CS250 (Schlötterer et al 1994) then sequenced as above.…”

“…Existing protocols for SHA homogenization disrupt individual bacteria, phytoplankton, invertebrate larvae, or other zooplankton by adding a chaotropic agent directly to the cryovial containing the sample and then heating (85°C for 5-10 min; Goffredi et al 2006;Jones et al 2008;Smith et al 2011). However, many fish eggs, including those from S. ocellatus, have a tough chorion membrane that protects the developing embryo.…”

“…The sandwich hybridization assay (SHA) is a rapid (ϳ1 h processing time) method of species or taxonomic group-specific identification that directly detects ribosomal RNA (rRNA) sequences and produces a quantifiable, colorimetric response. SHA has been used to identify and quantify harmful algal bloom species (Scholin et al 1996(Scholin et al , 1999Greenfield et al 2006Greenfield et al , 2008Ryan et al 2011;Doll et al 2014), bacteria (Preston et al 2009), zooplankton (Harvey et al 2012), and invertebrate larvae such as barnacles, green crabs, polychaetes, mussels, and starfish (Goffredi et al 2006;Jones et al 2008;Smith et al 2011). Similar to quantitative PCR (qPCR), SHA involves concentrating samples, breaking down cell membranes, and detecting target nucleic acid sequences.…”

“…Unlike qPCR, SHA does not entail extraction or amplification, detects rRNA rather than DNA, and quantifies enzymatic product absorbance, rather than labeled amplifier fluorescence. Additionally, SHA uses two rRNA-targeted oligonucleotides, a capture and a signal probe, that typically target small subunit or large subunit rRNA, although probes have also targeted the more variable internal transcribed spacer (ITS) region for invasive starfish (Smith et al 2011). To perform SHA, a sample homogenate is prepared by concentrating material on a filter and disrupting cell membranes using a chaotrope and heat to release nucleic acids.…”

Comparison of sandwich hybridization assay and quantitative PCR for the quantification of live and preserved cultures of Heterosigma akashiwo (Raphidophyceae)

“…2001). SHA has also been used to quantify non-HAB species such as marine invertebrates (Goffredi et al 2006;Smith et al 2011), bacteria (Preston et al 2009), and copepods (Harvey et al 2012).…”