Near real-time, autonomous detection of marine bacterioplankton on a coastal mooring in Monterey Bay, California, using rRNA-targeted DNA probes.
A sandwich hybridization assay (SHA) was developed to detect 16S rRNAs indicative of phylogenetically distinct groups of marine bacterioplankton in a 96-well plate format as well as low-density arrays printed on a membrane support. The arrays were used in a field-deployable instrument, the Environmental Sample Processor (ESP). The SHA employs a chaotropic buffer for both cell homogenization and hybridization, thus target sequences are captured directly from crude homogenates. Capture probes for seven of nine different bacterioplankton clades examined reacted specifically when challenged with target and non-target 16S rRNAs derived from in vitro transcribed 16S rRNA genes cloned from natural samples. Detection limits were between 0.10-1.98 and 4.43- 12.54 fmole ml(-1) homogenate for the 96-well plate and array SHA respectively. Arrays printed with five of the bacterioplankton-specific capture probes were deployed on the ESP in Monterey Bay, CA, twice in 2006 for a total of 25 days and also utilized in a laboratory time series study. Groups detected included marine alphaproteobacteria, SAR11, marine cyanobacteria, marine group I crenarchaea, and marine group II euryarchaea. To our knowledge this represents the first report of remote in situ DNA probe-based detection of marine bacterioplankton.
Preston
Christina M
CM
Monterey Bay Aquarium Research Institute, Moss Landing, CA 95039, USA. preston@mbari.org
Marin
Roman
R
Jensen
Scott D
SD
Feldman
Jason
J
Birch
James M
JM
Massion
Eugene I
EI
Delong
Edward F
EF
Suzuki
Marcelino
M
Wheeler
Kevin
K
Scholin
Christopher A
CA
D001105:Archaea / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D002140:California; D015342:DNA Probes / Q000235:genetics*; D046228:Microarray Analysis / Q000379:methods*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012623:Seawater / Q000382:microbiology*; D012680:Sensitivity and Specificity
D015342:DNA Probes; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S