Rapid identification of Vibrio vulnificus on nonselective media with an alkaline phosphatase-labeled oligonucleotide probe

Abstract

An oligonucleotide DNA probe (VYAP) was constructed from a portion of the Vibrio vulnificus cytolysin gene (hlyA) sequence and labeled with alkaline phosphatase covalently linked to the DNA. Control and environmental isolates probed with WAP showed an exact correlation with results obtained with a plasmid DNA probe (derived from pCVD702) previously described as having 100%/Y specificity and sensitivity for this organism. Identification of V. vulnificus strains was confirmed independently by analysis of the cellular fatty acid composition and by API 20E. Naturally occurring V. vulnificus bacteria were detected without enrichment or selective media by WAP in unseeded oyster homogenates and seawater collected from a single site in Chesapeake Bay during June at concentrations of 6 x 102 and 2 x 101 bacteria per ml, respectively. V. vulnificus bacteria were also enumerated by WAP in oysters seeded with known concentrations of bacteria and plated on nonselective medium. The WAP method provides a rapid, accurate means of identifying and enumerating V. vulnificus in seawater and oysters without the use of selective media or additional biochemical tests.

Detection of toxigenicVibrio choleraefrom environmental water samples by an enrichment broth cultivation-pit-stop semi-nested PCR procedure

“…The standard method for Vibrio detection involves isolation on a selective TCBS medium followed by a battery of biochemical and physiological tests. However, several problems are encountered with culturing methods, including the presence of viable but non-culturable cells, loss of viability of bacteria after collection, dif®culties in isolation from biocontaminated samples and the time required for culture and con®rmation, which can be several days (Wright et al 1993). To avoid these problems, different methods based on molecular biology techniques have been developed, with those based on DNA ampli®cation being the most rapid and sensitive (Garret et al 1993;Ramamurthy et al 1993).…”

“…To avoid these problems, different methods based on molecular biology techniques have been developed, with those based on DNA ampli®cation being the most rapid and sensitive (Garret et al 1993;Ramamurthy et al 1993). Nevertheless, ampli®ed products are seldom detected by direct visualization in ethidium bromide-stained agarose gels, but rather by Southern blot or dot-blot hybridization (Koch et al 1993;Wright et al 1993;Nair et al 1995). Although membrane hybridization is useful in research because it provides excellent sensitivity, these methods are generally time-consuming and labour-intensive.…”

First description of nonmotileVibrio vulnificusstrains virulent for eels

“…Several methods were also used to assay ODC and indole production tests (Altwegg et al, 1987;Smibert & Krieg, 1994), due to their importance for V. vulnificus biotyping. The final identification of bacterial isolates was performed by colony hybridization with a V. vulnificus-specific alkaline phosphatase-labeled DNA probe (DNA Technology, Aarhus, Denmark) directed against the cytolysin gene (hlyA) as previously described (Wright et al, 1993). Strains of Vibrio mimicus CECT 4218 T and V. vulnificus [CECT 529 T (biotype 1), CECT 4602 (biotype 2, serovar E) and CECT 5198 (biotype 2, serovar A)] were included for comparative purposes.…”

In vivogene expression of cold shock and other stress-related genes inVibrio vulnificusduring shellstock temperature control conditions in oysters

“…At various time intervals from t 0 to t 168 , V. vulnificus counts in oysters were enumerated using a species-specific oligonucleotide probe (vvhA) and colony blot hybridization procedure previously described by Wright et al (1993). Control filters were developed concurrently for all the time points.…”