OPTIMIZATION OF CONDITIONS FOR THE POLYMERASE CHAIN-REACTION AMPLIFICATION OF DNA FROM CULTURABLE AND NONCULTURABLE CELLS OF VIBRIO-VULNIFICUS

A series of 16 buffers, differing in pH and MgCl2, concentration were used to optimize the polymerase chain reaction (PCR) amplification of a 388 bp region of the hemolysin/cytolysin gene from cells of Vibrio v ulnificus present in both the culturable and nonculturable states. Bot h the opaque and translucent morphotypes were examined. Using whole ce ll lysates, we were able to obtain amplification of DNA from as few as 28.5 cells present in the viable but nonculturable state. With one ex ception, all buffers that produced amplification using culturable cell s also produced amplification using nonculturable cells. However, rega rdless of the buffer employed, 100 times more nonculturable cells than culturable cells were required to obtain a PCR product. Our data sugg est that caution should be exercised when employing PCR optimized agai nst culturable cells when this method is employed for the detection of nonculturable cells.