Experimental parameters affecting quantitative PCR of Artemia franciscana: a model for a marine zooplanktonic target in natural plankton samples

The brine shrimp Artemia franciscana was used as a model zooplankter to explore the range and accuracy of quantitative PCR (QPCR) in detecting a target species in plankton community DNA. Specific primers were designed in the 18S ribosomal RNA and mitochondrial cytochrome c oxidase subunit I genes and analyzed by TaqMan and SYBR Green I reporting systems. Assays were sensitive in detecting A. franciscana nauplii in DNA extractions from bulk plankton: a single nauplius was detected when added to 20 mg wet, packed plankton and distinguished from two added nauplii. Indeed, Artemia DNA diluted to 0.1 picograms per reaction (equivalent to 10(-5) nauplii) was detectable. Artemia franciscana was detected without coamplification of other plankton, alleviating concern for errors caused by the presence of similar organisms in the plankton community. A natural plankton DNA sample and purified herring sperm DNA inhibited PCR above 100 ng reaction(-1), but these samples could be diluted to eliminate inhibition. Because QPCR could detect 10-5 nauplii, dilution solves inhibition without sacrificing sensitivity. This sensitivity allows for analysis of a mass of plankton made large enough sufficient to include a target organism at low density. The newly hatched nauplius (DNA mass = 6. 1 ng) is a useful internal control in experiments examining plankton samples, providing a means of controlling for variations in DNA extraction and amplification efficiency in surveys for species of interest.