title: Underwater application of quantitative PCR on an ocean mooring.
abstract: The Environmental Sample Processor (ESP) is a device that allows for the underwater, autonomous application of DNA and protein probe array technologies as a means to remotely identify and quantify, in situ, marine microorganisms and substances they produce. Here, we added functionality to the ESP through the development and incorporation of a module capable of solid-phase nucleic acid extraction and quantitative PCR (qPCR). Samples collected by the instrument were homogenized in a chaotropic buffer compatible with direct detection of ribosomal RNA (rRNA) and nucleic acid purification. From a single sample, both an rRNA community profile and select gene abundances were ascertained. To illustrate this functionality, we focused on bacterioplankton commonly found along the central coast of California and that are known to vary in accordance with different oceanic conditions. DNA probe arrays targeting rRNA revealed the presence of 16S rRNA indicative of marine crenarchaea, SAR11 and marine cyanobacteria; in parallel, qPCR was used to detect 16S rRNA genes from the former two groups and the large subunit RuBisCo gene (rbcL) from Synecchococcus. The PCR-enabled ESP was deployed on a coastal mooring in Monterey Bay for 28 days during the spring-summer upwelling season. The distributions of the targeted bacterioplankon groups were as expected, with the exception of an increase in abundance of marine crenarchaea in anomalous nitrate-rich, low-salinity waters. The unexpected co-occurrence demonstrated the utility of the ESP in detecting novel events relative to previously described distributions of particular bacterioplankton groups. The ESP can easily be configured to detect and enumerate genes and gene products from a wide range of organisms. This study demonstrated for the first time that gene abundances could be assessed autonomously, underwater in near real-time and referenced against prevailing chemical, physical and bulk biological conditions.
authors.affiliation: Monterey Bay Aquarium Research Institute, Moss Landing, California, United States of America. preston@mbari.org
authors.forename: Christina M
authors.initials: CM
authors.lastname: Preston
authors.forename: Adeline
authors.initials: A
authors.lastname: Harris
authors.forename: John P
authors.initials: JP
authors.lastname: Ryan
authors.forename: Brent
authors.initials: B
authors.lastname: Roman
authors.forename: Roman
authors.initials: R
authors.lastname: Marin
authors.forename: Scott
authors.initials: S
authors.lastname: Jensen
authors.forename: Cheri
authors.initials: C
authors.lastname: Everlove
authors.forename: James
authors.initials: J
authors.lastname: Birch
authors.forename: John M
authors.initials: JM
authors.lastname: Dzenitis
authors.forename: Douglas
authors.initials: D
authors.lastname: Pargett
authors.forename: Masao
authors.initials: M
authors.lastname: Adachi
authors.forename: Kendra
authors.initials: K
authors.lastname: Turk
authors.forename: Jonathon P
authors.initials: JP
authors.lastname: Zehr
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Scholin
mesh_terms: D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D015342:DNA Probes; D007202:Indicators and Reagents; D008386:Marine Biology; D044085:Microfluidics; D009792:Oceans and Seas; D016133:Polymerase Chain Reaction / Q000379:methods*; D012335:RNA, Ribosomal / Q000235:genetics / Q000302:isolation & purification; D052616:Solid Phase Extraction
chemical_list: D017931:DNA Primers; D015342:DNA Probes; D007202:Indicators and Reagents; D012335:RNA, Ribosomal