DIRECT IDENTIFICATION OF VIBRIO VULNIFICUSIN CLINICAL SPECIMENS BY NESTED PCR

This study was performed to establish optimal nested PCR conditions an d a high-yield DNA extraction method for the direct identification of Vibrio vulnificus in clinical specimens. We designed two sets of prime rs targeting the V. vulnificus hemolysin/cytolysin gene. The target of the first primer set (P1-P2; sense, 5'-GAC-TAT-CGC-ATC-AAC-AAC-CG-3', and antiserase, 5'-AGG-TAG-CGA-GTA-TTA-CTG-CC-3', respectively) is a 704-bp DNA fragment. The second set (P3-P4; sense, 5'-GCT-ATT-TCA-CCG- CCG-CTC-AC-3', and antisense, 5'-CCG-CAG-AGC-CGT-AAA-CCG-AA-3', respec tively) amplifies an internal 222-bp DNA fragment. We developed a dire ct DNA extraction method that involved boiling the specimen pellet in a 1 mM EDTA-0.5% Triton X-100 solution. The new DNA extraction method was more sensitive and reproducible than other conventional methods. T he DNA extraction method guaranteed sensitivity as well, even when V. vulnificus cells were mixed with other bacteria such as Escherichia co li or Staphylococcus aureus. The nested PCR method could detect as lit tle as 1 fg of chromosomal DNA and single CFU of V, vulnificus. We app lied the nested PCR protocol to a total of 39 serum specimens and bull a aspirates from septicemic patients. Seventeen (94.4%) of the 18 V. v ulnificus culture-positive specimens were positive by the nested PCR, Eight (42.1%) of the 19 culture-negative samples gave positive nested PCR results.