A 96-Well Plate Format for Detection of Marine Zooplankton with the Sandwich Hybridization Assay

  • Julio B. J. Harvey
Part of the Methods in Molecular Biology book series (MIMB, volume 1128)


The sandwich hybridization assay (SHA) is a ribosomal RNA (rRNA) targeted molecular method used to detect specific target organisms from diverse communities found in environmental water samples. This sensitive, robust assay is particularly useful for detecting zooplankton, including copepod grazers or reproductive propagules from broadcast spawning invertebrates. Herein, I describe the most basic application of this flexible methodology—a 96-well plate format for analysis of water samples in the laboratory. A microarray format SHA is also available and uses the same basic chemistry for remote, robotically mediated, in situ target detection. Traditionally produced only in the laboratory, preassembled SHA reagents and consumables are now also available for purchase.

Key words

Sandwich hybridization assay Molecular probes rDNA rRNA Invertebrate larvae Copepods Invasive species detection 



This work was supported by and conducted at the Monterey Bay Aquarium Research Institute. Sincere thanks to R. Marin III, T. Hurford, C. Melançon, C. Preston, N. Alvarado, C. A. Scholin, and R. C. Vrijenhoek for their invaluable advice and assistance.


  1. 1.
    Greenfield DI et al (2006) 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. Limnol Oceanogr Meth 4:426–435CrossRefGoogle Scholar
  2. 2.
    Goffredi SK et al (2006) Molecular detection of marine invertebrate larvae. Mar Biotechnol 8:149–160PubMedCrossRefGoogle Scholar
  3. 3.
    Haywood AJ et al (2007) Molecular detection of the brevetoxin-producing dinoflagellate Karenia brevis and closely related species using rRNA-targeted probes and a semiautomated sandwich hybridization assay. J Phycol 43:1271–1286CrossRefGoogle Scholar
  4. 4.
    Scholin CA et al (1999) DNA probes and a receptor-binding assay for detection of Pseudo-nitzschia (Bacillariophyceae) species and domoic acid activity in cultured and natural samples. J Phycol 35:1356–1367Google Scholar
  5. 5.
    Marin R III, Scholin CA (2010) Toxic algal detection using rRNA-target probes in a semi-automated sandwich hybridization format. In: Karlson B, Cusack C, Bresnan E (eds) Microscopic and molecular methods for quantitative phytoplankton analysis. Intergovernmental Oceanographic Commission of UNESCO, Paris, p 110Google Scholar
  6. 6.
    Ayers K et al (2005) International accreditation of sandwich hybridisation assay format DNA probes for micro-algae. N Z J Mar Freshwater Res 39:1225–1231CrossRefGoogle Scholar
  7. 7.
    Tyrrell JV et al (2001) Detection and enumeration of Heterosigma akashiwo and Fibrocapsa japonica (Raphidophyceae) using rRNA-targeted oligonucleotide probes. Phycologia 40:457–467CrossRefGoogle Scholar
  8. 8.
    Mikulski CM et al (2008) Development and field application of rRNA-targeted probes for the detection of Cochlodinium polykrikoides Margalef in Korean coastal waters using whole cell and sandwich hybridization formats. Harmful Algae 7:347–359CrossRefGoogle Scholar
  9. 9.
    Rhodes LL, Adamson J, Scholin C (2000) Pseudo-nitzschia multistriata (Bacillariophyceae) in New Zealand. N Z J Mar Freshwater Res 34:463–467CrossRefGoogle Scholar
  10. 10.
    Preston CM et al (2009) Near real-time, autonomous detection of marine bacterioplankton on a coastal mooring in Monterey Bay, California, using rRNA-targeted DNA probes. Environ Microbiol 11:1168–1180PubMedCrossRefGoogle Scholar
  11. 11.
    Jones WJ et al (2008) A robotic molecular method for in situ detection of marine invertebrate larvae. Molecular Ecology Resources 8:540–550PubMedCrossRefGoogle Scholar
  12. 12.
    Doucette GJ et al (2009) Remote, subsurface detection of the algal toxin domoic acid onboard the environmental sample processor: assay development and field trials. Harmful Algae 8:880–888CrossRefGoogle Scholar
  13. 13.
    Scholin C et al (2009) Remote detection of marine microbes, small invertebrates, harmful algae, and biotoxins using the environmental sample processor (ESP). Oceanography 22:158–167CrossRefGoogle Scholar
  14. 14.
    Scholin CA et al. (2001) Aquatic autosampler device. US patent 6187530, USAGoogle Scholar
  15. 15.
    Greenfield D et al (2008) Field applications of the second-generation environmental sample processor (ESP) for remote detection of harmful algae: 2006–2007. Limnol Oceanogr Meth 6:667–679CrossRefGoogle Scholar
  16. 16.
    Scholin CA et al (1996) Identification of Pseudo-nitzschia australis using rRNA-targeted probes in whole cell and sandwich hybridization formats. Phycologia 35:190–197CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Julio B. J. Harvey
    • 1
  1. 1.Monterey Bay Aquarium Research InstituteMoss LandingUSA

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