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Applied and Environmental Microbiology, July 2008, p. 4336-4345, Vol. 74, No. 14
0099-2240/08/$08.00+0     doi:10.1128/AEM.02131-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Development of a Denaturing High-Performance Liquid Chromatography Method for Detection of Protist Parasites of Metazoans ^,

Skidaway Institute of Oceanography, 10 Ocean Science Circle, Savannah, Georgia 31411,¹ Department of Biology, University of Bergen, P.O. Box 7800, N-5020 Bergen, Norway²

Received 17 September 2007/ Accepted 12 May 2008

Increasingly, diseases of marine organisms are recognized as significant biotic factors affecting ecosystem health. However, the responsible disease agents are often unknown and the discovery and description of novel parasites most often rely on morphological descriptions made by highly trained specialists. Here, we describe a new approach for parasite discovery, utilizing denaturing high-performance liquid chromatography (DHPLC) reverse-phase ion-paring technology. Systematic investigations of major DHPLC variables, including temperature, gradient conditions, and target amplicon characteristics were conducted to develop a mechanistic understanding of DNA fragment separation by DHPLC. As a model system, 18S rRNA genes from the blue crab (Callinectes sapidus) and a parasitic dinoflagellate Hematodinium sp. were used. Binding of 18S rRNA gene PCR amplicons to the DNA separation column in the presence of triethylammonium acetate (TEAA) was inversely correlated with temperature and could be predicted based on the estimated DNA helicity of the PCR amplicon. Amplicons of up to 498 bp were resolved as single chromatographic peaks if they had high (>95%) DNA helicity. Amplicons that differed by as few as 2 bp could be resolved. Separation of 18S rRNA gene PCR amplicons was optimized by simultaneous manipulation of both temperature and solvent gradients. The optimal conditions included targeting regions of high DNA helicity (>95%), temperatures in the range of 57 to 63°C, and a linear acetonitrile gradient from 13.75 to 17.5% acetonitrile in 0.1 M TEAA (55 to 70% buffer B) over a 9-min period. Under these conditions, amplicons from a variety of parasites and their hosts can be separated and detected by DHPLC.

Published ahead of print on 23 May 2008.

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This article has been cited by other articles:

• Troedsson, C., Lee, R. F., Walters, T., Stokes, V., Brinkley, K., Naegele, V., Frischer, M. E. (2008). Detection and Discovery of Crustacean Parasites in Blue Crabs (Callinectes sapidus) by Using 18S rRNA Gene-Targeted Denaturing High-Performance Liquid Chromatography. Appl. Environ. Microbiol. 74: 4346-4353 [Abstract] [Full Text]