|Graczyk, T - JHUSHPH|
|Kerby, S - USAMRIID|
|Lewis, E - NOAA|
Submitted to: Journal of Shellfish Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 18, 2000
Publication Date: N/A
Interpretive Summary: Because of the slimy nature of their tissues, shellfish such as oysters can be difficult to assay for food-borne pathogens, using molecular biology techniques such as the polymerase chain reaction (PCR). It is usually necessary to perform a lengthy and complication procedure to obtain pure nucleic acids from the oysters. In this paper, the authors evaluated more rapid, and less expensive, methods to prepare oysters for PCR: phase lock gels, and two kinds of filter paper techniques. The phase lock gels and filter papers proved to be easy to use, quick to perform, and yielded nucleic acids suitable for PCR. These techniques can be used to screen large numbers of shellfish samples, for molecular genetics and population biology studies.
Technical Abstract: Performing successful PCR assays on oysters and other shellfish requires obtaining purified nucleic acids, free of inhibitory substances, from the tissues, hemolymph, and secretions of these animals. We investigated the use of three methods to extract nucleic acids from gill washings and hemolymph from Eastern oysters, Crassostrea virginica. They were: phenol-chloroform-isoamyl alcohol (pci); FTA paper; and Isocode paper. All methods yielded nucleic acids capable of serving as templates for oyster small subunit ribosomal RNA (SSU rRNA) PCR. The pci method took under 2 hrs to perform and provided template suitable for detecting pathogen DNA by nested PCR. The FTA and Isocode paper methods were inexpensive, used fewer and less hazardous reagents, and took less than 1 hour to perform. The paper-based methods are also readily adaptable for use in high-throughput format.