Detection of Vibrio vulnificus biotypes 1 and 2 in eels and oysters by PCR amplification.
DNA extraction procedures and PCR conditions to detect Vibrio vulnificus cells naturally occurring in oysters were developed. In addition, PCR amplification of V. vulnificus from oysters seeded with biotype 1 cells was demonstrated. By the methods described, V. vulnificus cells on a medium (colistin-polymyxin B-cellobiose agar) selective for this pathogen were detectable in oysters harvested in January and March, containing no culturable cells (< 67 CFU/g), as well as in oysters harvested in May and June, containing culturable cells. It was possible to complete DNA extraction, PCR, and gel electrophoresis within 10 h by using the protocol described for oysters. V. vulnificus biotype 2 cells were also detected in eel tissues that had been infected with this strain and subsequently preserved in formalin. The protocol used for detection of V. vulnificus cells in eels required less than 5 h to complete. Optimum MgCl2 concentrations for the PCR of V. vulnificus from oysters and eels were different, although the same primer pair was used for both. This is the first report on the detection of cells of V. vulnificus naturally present in shellfish and represents a potentially powerful method for monitoring this important human and eel pathogen.
Coleman
S S
SS
Department of Biology, University of North Carolina at Charlotte 28223, USA.
Melanson
D M
DM
Biosca
E G
EG
Oliver
J D
JD
D000818:Animals; D001431:Bacteriological Techniques; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004524:Eels / Q000382:microbiology*; D005069:Evaluation Studies as Topic; D006801:Humans; D008969:Molecular Sequence Data; D010125:Ostreidae / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D014733:Vibrio / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D014774:Virulence / Q000235:genetics
D017931:DNA Primers; D004269:DNA, Bacterial