A PCR-ELISA Method for Direct Detection of the Oyster Pathogen Haplosporidium nelsoni |
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| Authors: | Yuan-Tih Ko Marion Man-Ying Chan Susan E Ford Dunne Fong |
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| Institution: | (1) Department of Nutrition, China Medical College, Taichung, Taiwan 40421, TW;(2) Department of Microbiology and Immunology, Temple University School of Medicine, 3400 N. Broad St., Philadelphia, PA 19140, U.S.A., US;(3) Haskin Shellfish Research Laboratory, Rutgers, The State University of New Jersey, Port Norris, NJ 08349, U.S.A., US;(4) Division of Life Sciences, Nelson Biological Laboratories, Rutgers, The State University of New Jersey, 604 Allison Road, Piscataway, New Jersey 08854, U.S.A., US |
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| Abstract: | A rapid method, utilizing both polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), was developed
for detection of oyster MSX disease. The technique included using Haplosporidium nelsoni pathogen-specific PCR primers (based on ribosomal RNA genes), a Chelex resin (for rapid DNA extraction from oyster mantle
tissues), and cloned H. nelsoni rRNA plasmid DNA (for use as a capture probe). Digoxigenin was incorporated into the pathogen-specific PCR products, which
were captured by the coated probe in a fast hybridization reaction and then detected by ELISA. The sensitivity of PCR amplification
on cloned plasmid DNA was 10 fg for detection by stained agarose gel, and increased to 0.01 fg for ELISA. Positive signals
were observed in infected oysters using the PCR-ELISA technique. This method may be applicable to early detection of infection.
Received April 14, 1998; accepted September 30, 1998. |
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| Keywords: | :Crassostrea virginica Haplosporidium nelsoni rRNA PCR hybridization ELISA |
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