Advertisement

Marine Biotechnology

, Volume 2, Issue 5, pp 456–465 | Cite as

Use of Competitive PCR to Detect and Quantify Haplosporidium nelsoni Infection (MSX disease) in the Eastern Oyster (Crassostrea virginica)

  • J. Michael  Day
  • Dean E.  Franklin
  • Bonnie L.  Brown

Abstract:

This study was undertaken to develop a quantitative polymerase chain reaction assay that would improve the utility of PCR for detecting Haplosporidium nelsoni (MSX), a serious parasite of the eastern oyster Crassostrea virginica. A competitive PCR sequence was generated from the H. nelsoni small subunit ribosomal DNA fragment, originally described by Stokes and colleagues, that was amplified by the same PCR primers and had similar amplification performance. Assays performed using competitor dilutions ranging from 0.05 to 500 pg/μl DNA were used to test oyster samples designated using histological techniques as having ``light'' or ``heavy'' MSX infections. Visual diagnoses were confirmed equally well with three methods: densitometry of ethidium-bromide-stained agarose, densitometry of SYBRGreen-stained polyacrylamide gels, and analysis by GeneScan 3.0 of fluorescent products detected in ultrathin gels. Oysters diagnosed as negative for MSX tested as negative or light by PCR. Oysters with light MSX infections generally had less than 5 pg/μl infectious DNA. Oysters with heavy infections generally corresponded to 5 pg/μl or greater competitor dilutions.

Key words: PCR quantitative-competitive PCR (QC-PCR), Haplosporidium nelsoni, MSX, Crassostrea virginica, eastern oyster. 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Copyright information

© Springer-Verlag New York Inc. 2000

Authors and Affiliations

  • J. Michael  Day
    • 1
  • Dean E.  Franklin
    • 1
  • Bonnie L.  Brown
    • 1
  1. 1.Department of Biological Sciences, Virginia Commonwealth University, Richmond, VA 23284, U.S.A.US

Personalised recommendations