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.
Highlights
Modern molecular biological techniques have revolutionized our understanding of the diversity, function and community structure of marine microorganisms
One of the techniques commonly employed in this regard is quantitative PCR which is used to detect and enumerate unique nucleic acid sequences indicative of specific organisms, functional genes and other genetic markers in samples collected from a wide array of environments [5,6,7,8,9]
We describe the development and application of methods that utilize a reusable solid phase nucleic acid extraction system and 2-channel real-time PCR module that was integrated with the Microfluidic Block (MFB) and Environmental Sample Processor (ESP)
Summary
Modern molecular biological techniques have revolutionized our understanding of the diversity, function and community structure of marine microorganisms (for review see [1,2,3]). One of the techniques commonly employed in this regard is quantitative PCR (qPCR; [4]) which is used to detect and enumerate unique nucleic acid sequences indicative of specific organisms, functional genes and other genetic markers in samples collected from a wide array of environments [5,6,7,8,9]. For the vast majority of ocean science and resource management applications of qPCR, discrete samples are collected and preserved in the field, returned to a laboratory where they are generally processed in batch mode many hours, days, or months later to reveal targets of interest. Is qPCR applied in the field, adaptively, in support of ocean research and monitoring. Opportunities to alter sample acquisition schemes or adapt experimental procedures based on results of qPCR assays are limited
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