Title
Monterey Bay Aquarium Research Institute
Marine Botany

Pseudo-nitzschia Identification

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Species Identification

Traditionally, identification of species within Pseudo-nitzschia has been accomplished through scanning electron microscopy. This is, however, an arduous and time-consuming process that involves an expert taxonomist. More efficient identification methods have been adopted, especially in light of the necessity to prevent Amnesic Shellfish Poisoning (see Toxin page) outbreaks. Two methods have been developed that expedite this process: the Whole Cell Hybridization Method and the Sandwich Hybridization Method. Both methods target ribosomal RNA (rRNA) since each species of Pseudo-nitzschia contains unique sequences.

Whole Cell Hybridization Method

  • preserved cells (kept in ethanol saline solution that does not disrupt cells)are incubated with a fluorescently labeled, rRNA-targeted oligonucleotide
  • cells are rinsed to remove excess/unbound probe
  • those cells that retain probe are visualized via epifluorescence microscopy
  • advantage: it is faster than the SEM method and also visualizes intact (preserved) cells
  • disadvantage: it cannot be automated and is thus still labor intensive (3 hours process)

 

Sandwich Hybridization Method

  • hybridization of species-specific capture probe to complementary rRNA sequences (this immobilizes target sequences in lysate)
  • hybridization of biotinylated signal probe to target rRNA
  • adsorption of avidin-enzyme conjugate to the signal probe
  • visualization of capture probe-target-signal probe sandwiches by an enzyme driven color reaction
  • advantage: fast (30 minutes), can be automated
  • disadvantage: cells must be lysed prior to process (whole cell comparison impossible)

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copyright Jennifer Shin 1999.

Last updated: Feb. 05, 2009