Direct Identification of Vibrio vulnificus in Clinical Specimens by Nested PCR |
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| Authors: | Shee Eun Lee Soo Young Kim Sei Jong Kim Hyun Soo Kim Jong Hee Shin Sang Ho Choi Sun Sik Chung Joon Haeng Rhee |
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| Affiliation: | Department of Microbiology,1. Department of Internal Medicine,3. and Department of Clinical Pathology,4. Chonnam National University Medical School, and Institute of Medical Sciences,2. Chonnam National University, Kwangju 501-190, and Department of Food Science and Technology, Chonnam National University, Kwangju 500-757,5. Republic of Korea |
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| Abstract: | This study was performed to establish optimal nested PCR conditions and a high-yield DNA extraction method for the direct identification of Vibrio vulnificus in clinical specimens. We designed two sets of primers 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 antisense, 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′, respectively) amplifies an internal 222-bp DNA fragment. We developed a direct 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. The DNA extraction method guaranteed sensitivity as well, even when V. vulnificus cells were mixed with other bacteria such as Escherichia coli or Staphylococcus aureus. The nested PCR method could detect as little as 1 fg of chromosomal DNA and single CFU of V. vulnificus. We applied the nested PCR protocol to a total of 39 serum specimens and bulla aspirates from septicemic patients. Seventeen (94.4%) of the 18 V. vulnificus culture-positive specimens were positive by the nested PCR. Eight (42.1%) of the 19 culture-negative samples gave positive nested PCR results. |
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