DETECTION OF VIBRIO-VULNIFICUS BIOTYPE-1 AND BIOTYPE-2 IN EELS AND OYSTERS BY PCR AMPLIFICATION

DNA extraction procedures and PCR conditions to detect Vibrio vulnific us cells naturally occurring in oysters were developed. In addition, P CR amplification of V. vulnificus from oysters seeded with biotype 1 c ells was demonstrated. By the methods described, V. vulnificus cells o n a medium (colistin-polymyxin B-cellobiose agar) selective for this p athogen were detectable in oysters harvested in January and March, con taining no culturable cells (<67 CFU/g), as well as in oysters harvest ed in May and June, containing culturable cells. It was possible to co mplete DNA extraction, PCR, and gel electrophoresis within 10 h by usi ng the protocol described for oysters. V. vulnificus biotype 2 cells w ere also detected in eel tissues that had been infected with this stra in and subsequently preserved in formalin. The protocol used for detec tion of V. vulnificus cells in eels required less than 5 h to complete . Optimum MgCl2 concentrations for the PCR of V. vulnificus from oyste rs 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. vulnif icus naturally present in shellfish and represents a potentially power ful method for monitoring this important human and eel pathogen.