Publication Abstract

Despite advancements in the area of in-field detection of pathogens in aquatic organisms, limited research has evaluated point-of-need DNA extraction methods. This is a critical step in the application of any field deployable molecular assays. This is especially important to applications that use PCR, because carry over of contaminants causes inhibition and false negative results. To address this, we compared the performance of nine rapid DNA extraction methods with a control laboratory method (QIAMP DNA mini kit, QIAGEN). DNA was extracted from fresh, frozen and ethanol-fixed mantle and digestive tissues of Pacific oysters (Magallana gigas). Each assay extraction performance was assessed by measuring DNA quantity and contaminants using fluorescence and spectrophotometric methods respectively. Additionally, PCR amplification of the host cytochrome oxidase subunit 1 gene (COX1) was performed to assess the presence of inhibitors. The QuickExtract™ DNA extraction solution (QEDES, Lucigen) yielded the highest DNA quantity and carried over minimal contaminants, resulting in low PCR inhibition. In addition the suitability of QEDES for detection of oyster pathogens was assessed, using M. gigas samples infected with Ostreid herpesvirus 1 and Vibrio aestuarianus and European flat oysters (Ostrea edulis) infected with Bonamia ostreae. Conventional and qPCR were used to assess performance. In each instance, the QEDES successfully identified the presence of the pathogens tested.

Rose Kerr, Matt Edwards, Robert Hatfield and Frederico Batista.