Experimental parameters affecting quantitative PCR of Artemia franciscana: a model for a marine zooplanktonic target in natural plankton samples
Limnol. Oceanogr. Methods 8:337-347 (2010) | DOI: 10.4319/lom.2010.8.337
ABSTRACT: 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 105 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 reaction1, but these samples could be diluted to eliminate inhibition. Because QPCR could detect 105 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.