Sample preparation methods for quantitative detection of DNA by molecular assays and marine biosensors |
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| Authors: | Annie M Cox Kelly D Goodwin |
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| Institution: | 1. National Oceanic & Atmospheric Administration (NOAA), Northwest Fisheries Science Center, La Jolla, CA 92037, USA;2. National Oceanic & Atmospheric Administration (NOAA), Atlantic Oceanographic & Meteorological Laboratories (AOML), Miami, FL 33149, USA |
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| Abstract: | The need for quantitative molecular methods is growing in environmental, food, and medical fields but is hindered by low and variable DNA extraction and by co-extraction of PCR inhibitors. DNA extracts from Enterococcus faecium, seawater, and seawater spiked with E. faecium and Vibrio parahaemolyticus were tested by qPCR for target recovery and inhibition. Conventional and novel methods were tested, including Synchronous Coefficient of Drag Alteration (SCODA) and lysis and purification systems used on an automated genetic sensor (the Environmental Sample Processor, ESP). Variable qPCR target recovery and inhibition were measured, significantly affecting target quantification. An aggressive lysis method that utilized chemical, enzymatic, and mechanical disruption enhanced target recovery compared to commercial kit protocols. SCODA purification did not show marked improvement over commercial spin columns. Overall, data suggested a general need to improve sample preparation and to accurately assess and account for DNA recovery and inhibition in qPCR applications. |
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| Keywords: | Sample preparation Inhibition DNA extraction SCODA Environmental sample processor ESP qPCR |
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