Use of competitive PCR to detect and quantify Haplosporidium nelsoni infection (MSX disease) in the eastern oyster (Crassostrea virginica)

This study was undertaken to develop a quantitative polymerase chain reacti on assay that would improve the utility of PCR for detecting Haplosporidium nelsoni (MSX), a serious parasite of the eastern oyster Crassostrea virgin ica. A competitive PCR sequence was generated from the H. nelsoni small sub unit ribosomal DNA fragment, originally described by Stokes and colleagues, that was amplified by the same PCR primers and had similar amplification p erformance. Assays performed using competitor dilutions ranging from 0.05 t o 500 pg/mul DNA were used to test oyster samples designated using histolog ical techniques as having "light" or "heavy" MSX infections. Visual diagnos es 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 ultr athin gels. Oysters diagnosed as negative for MSX tested as negative or lig ht by PCR. Oysters with light MSX infections generally had less than 5 pg/m ul infectious DNA. Oysters with heavy infections generally corresponded to 5 pg/mul or greater competitor dilutions.