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Experimental parameters affecting quantitative PCR of Artemia franciscana : a model for a marine zooplanktonic target in natural plankton samples
Author(s) -
Mackie Joshua A.,
Geller Jonathan
Publication year - 2010
Publication title -
limnology and oceanography: methods
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.898
H-Index - 72
ISSN - 1541-5856
DOI - 10.4319/lom.2010.8.337
Subject(s) - plankton , biology , brine shrimp , dna extraction , dna , taqman , zoology , chromatography , polymerase chain reaction , ecology , biochemistry , gene , chemistry
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.