Comparison of Conventional, Nested, and Real-Time PCR Assays for Rapid and Accurate Detection of Vibrio vulnificus

Abstract

We conducted a prospective study to target toxR in the blood of patients with skin and soft tissue infections who were admitted to four tertiary hospitals to assess the clinical usefulness of real-time quantitative PCR (Q-PCR) as a diagnostic technique. We performed conventional PCR (C-PCR), nested PCR (N-PCR), and Q-PCR assays and compared the results to those obtained using the "gold standard" of microbiological culture. The lower detection limit for the Q-PCR assay was 5 ؋ 10 0 copies/l. By use of blood samples of patients with skin and soft tissue infections, the sensitivities of the C-PCR and N-PCR assays against the target toxR gene of V. vulnificus as diagnostic tools were determined to be 45% and 86%, respectively. The C-PCR and N-PCR assays had specificities of 100% and 73%, respectively. When we adopted a crossing-point (cp) cutoff value of <38 cp as a positive result, the Q-PCR assay had 100% sensitivity and specificity. Q-PCR to detect V. vulnificus-specific genes is not only the most sensitive and specific of the techniques but also the most rapid diagnostic method. Therefore, the appropriate application of the Q-PCR assay using blood is useful for the rapid diagnosis and subsequent treatment of V. vulnificus sepsis.Vibrio vulnificus can cause severe and life-threatening disease in those who eat contaminated seafood or have a wound that is exposed to seawater (2,11,15,24). The disease develops rapidly and mortality is high. Hence, these patients require rapid diagnosis and subsequent treatment. Microbiological culture methods for the identification of causative organisms take several days; they are time-consuming and laborious but have good specificity (13). PCR assays have proven useful for early diagnosis. Conventional PCR (C-PCR) has been used to detect specific target genes in various microorganisms (5, 6, 13). Nested PCR (N-PCR) was developed to improve sensitivity but can give erroneous positive results due to DNA contamination (1). Multiplex PCR has the advantage of detecting several target genes at the same time, but it is time-consuming and laborious like 14). Real-time quantitative PCR (Q-PCR) can detect V. vulnificus-specific genes within 2 h (4, 15); there is no agarose gel-loading step (23), and the assay is not laborious and has high sensitivity and specificity (22).Up to now there has been little comparative evaluation of these three PCR methods, namely, C-PCR, N-PCR, and Q-PCR, for targeting V. vulnificus-specific genes. The toxR gene is known as a gene encoding a transmembrane DNA binding regulatory protein in Vibrio species. The partial sequences of toxR differ among Vibrio species. The difference in toxR sequences among Vibrio species has been used as an effective marker for the identification of Vibrio species (22). To assess the clinical usefulness of Q-PCR as a diagnostic technique, we conducted a prospective study targeting the toxR gene of V. vulnificus in blood samples of patients with skin and soft tissue infections who were admitted to four tertiary-care hospitals. We carr...

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Vibrio vulnificus DNA Load and Mortality

“…Real-time PCR technology enables quantification of microorganisms (9,16). An association between disease severity/mortality and bacterial DNA load measured by quantitative real-time PCR has been demonstrated for Neisseria meningitidis and Staphylococcus aureus infections (2, 6), but there has been no such study reported for patients with Vibrio septicemia.…”

“…AF170883). The primers used were tox-130 (T GTTCGGTTGAGCGCATTAA) and tox-200 (GCTTCAGA GCTGCGTCATTC), and the probe was (6-carboxyfluoresc ein-CGCTCCTGTCAGATTCAACCAACAACG-Black Hole Quencher-1) (9,14). V. vulnificus infection was confirmed by identifying V. vulnificus in blood, skin, or soft tissue.…”

“…Blood sera of 22 of the V. vulnificus-infected patients in our previous study (9) were included in this study. Blood, skin, and soft tissues were cultured on admission.…”

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