Development of quantitative, real-time PCR probes for the automated detection of toxic dinoflagellates in seawater samples

Increasing knowledge of the ribosomal RNA gene sequences of harmful dinoflagellates has enabled the development of molecular probe- based detection technologies to progress from lab-based research tools to commercial products used for routine screening of cultured and natural samples.

Early work concentrated on fluorescently-labelled antibody and lectin probes, but current probe design centers on the use of RNA- or DNA-targeted probes. The most common probe formats have so far been FITC-labelled fluorescent in situ hybridisation (FISH) using whole cells, and sandwich hybridisation using crude cell lysates.

FISH probe technology is mostly in the public domain and inexpensive to run provided access to a fluorescent microscope is available. Thus they are the choice method for several commercial laboratories, including the Cawthron Institute, in New Zealand.

Recent advaces in the use of real-time, quantitative PCR (qPCR), have been recently applied to the detection of toxic algae. qPCR probes have been designed to detect several fish-killing dinoflagellates, and probes for species causing paralytic shellfish poisoning (PSP) are in development. qPCR technology using dual-labelled probes allows the detection of as little as ten copies of a dinoflagellate gene which is usually present in 1,000-4,000 copies per cell. Additionally, run times can be shorten to as little as 1 s denaturation and 1 s annealing/extension, completing the run in under 40 min.

The use of molecular probes in algal detection has progressed from a lab-based tool to discriminate morphologically similar species to a commercial technique used in routine algal monitoring.

Aditionally, the qPCR techniques developed primarily for use in human health have been successfully adapted for use in the quantitative detection of harmful algae in water samples. The main potential of qPCR techniques is being realised as this technology is combined with the use of automated sampling and handling systems, which enable hight throughput of samples. The development fully-automated systems which collect water samples, lyse the cell pellet, and carry our qPCR on the lysate, sending the results back to the lab in real-time, allow access to data of population trends of toxic species that has hitherto been inaccessible, and can provide early warning of developing blooms below the detection threshold of microscopy-based systems.

qPCR run of serial dilutions of a known concentration standard DNA of the highly toxic Karenia brevisulcata dinoflagellate.

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qPCR runs for the detection of toxic dinoflagellates can be reduced to a total time of only 40 minutes while keeping excellent consistency between replicates.

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