Sample preparation methods for quantitative detection of DNA by molecular assays and marine biosensors.

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.

journal_name

Mar Pollut Bull

authors

Cox AM,Goodwin KD

doi

10.1016/j.marpolbul.2013.06.006

subject

Has Abstract

pub_date

2013-08-15 00:00:00

pages

47-56

issue

1

eissn

0025-326X

issn

1879-3363

pii

S0025-326X(13)00296-8

journal_volume

73

pub_type

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