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

• Published in: Applied and Environmental Microbiology 1993-02, Vol.59 (2), p.541-546
• Main Authors: Wright, A.C. (University of Maryland School of Medicine, Baltimore, MD), Miceli, G.A, Landry, W.L, Christy, J.B, Watkins, W.D, Morris, J.G. Jr
• Format: Article
• Language: English
• Subjects: ADN; Alkaline Phosphatase; ANALISIS BIOLOGICO; ANALYSE BIOLOGIQUE; Animals; BACTERIA; Bacteriological methods and techniques used in bacteriology; Bacteriology; Base Sequence; BIODEGRADACION; BIODEGRADATION; Biological and medical sciences; Biotechnology; Cellular biology; Colony Count, Microbial; CONTAMINACION; CONTAMINATION; CONTEO DE CELULAS; Deoxyribonucleic acid; DNA; FOSFATASA ALCALINA; Fundamental and applied biological sciences. Psychology; HIBRIDACION DE ADN; HUITRE; HYBRIDATION D'ADN; IDENTIFICACION; IDENTIFICATION; INFECCION; INFECTION; MEDIO DE CULTIVO; Microbiology; MILIEU DE CULTURE; Molecular Sequence Data; NUMERATION CELLULAIRE; Oligonucleotide Probes - chemistry; OSTRA; Ostreidae - microbiology; PHOSPHATASE ALCALINE; Seawater; SECUENCIA NUCLEICA; SEQUENCE NUCLEIQUE; TOXINAS; TOXINE; VIBRIO; Vibrio - classification; Vibrio - growth & development; Vibrio - isolation & purification; Vibrio vulnificus; Water Microbiology
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/AEM.59.2.541-546.1993

An oligonucleotide DNA probe (VVAP) 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 VVAP showed an exact correlation with results obtained with a plasmid DNA probe (derived from pCVD702) previously described as having 100% 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 VVAP in unseeded oyster homogenates and seawater collected from a single site in Chesapeake Bay during June at concentrations of 6 X 10(2) and 2 X 10(1) bacteria per ml, respectively. V. vulnificus bacteria were also enumerated by VVAP in oysters seeded with known concentrations of bacteria and plated on nonselective medium. The VVAP 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