Rapid detection of Vibrio species using liquid microsphere arrays and real-time PCR targeting the ftsZ locus

Abstract

The development of rapid and sensitive molecular techniques for the detection of Vibrio species would be useful for the surveillance of sporadic infections and management of major outbreaks. Comparative sequence analysis of the ftsZ gene in the predominant Vibrio species that cause human disease revealed distinct alleles for each examined species, including Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus. Light Upon eXtension (LUX) real-time PCR assays were developed to target these species-specific polymorphisms, and were successful in rapidly differentiating the major pathogenic Vibrio species. Luminex liquid microsphere array technology was used to develop a comprehensive assay capable of simultaneously detecting V. cholerae, V. parahaemolyticus and V. vulnificus. These assays permitted the identification of a presumptive V. parahaemolyticus isolate as Vibrio alginolyticus, which was verified using additional molecular characterization. INTRODUCTIONPathogenic species of the genus Vibrio pose a considerable public health threat as the causative agents of both sporadic and epidemic human infections. Cholera, caused by Vibrio cholerae, continues to spread globally in a seventh pandemic (O1 El Tor biotype), and the emergence of a non-O1 serogroup (O139 Bengal) has led to a new pandemic (Sack et al., 2004;Faruque & Mekalanos, 2003). Unlike the watery diarrhoeal disease caused by V. cholerae, infection with Vibrio parahaemolyticus usually results in a self-limiting gastroenteritis with inflammatory diarrhoea and, in rare cases, septicaemia (Janda et al., 1988). Opportunistic infections with Vibrio vulnificus can cause severe wound infections (Oliver, 2005) and fulminant septicaemia, with highly virulent strains causing extensive host-tissue damage and producing mortality rates of up to 60 % (Linkous & Oliver, 1999). The threat posed by pathogenic Vibrio species has been highlighted by recent natural disasters, in which a number of survivors were infected in the aftermath of Hurricanes Katrina and Rita on the gulf coast of the USA (Centers for Disease Control, 2005Control, , 2006, and the Indian Ocean tsunami (Lim, 2005). Fortunately, major outbreaks did not occur, but these disasters have served as an impetus to develop rapid and sensitive molecular techniques that can be easily deployed to identify pathogenic Vibrio species during a public health emergency.Traditionally, identification of Vibrio spp. has consisted of isolation on selective agar medium followed by biochemical and serological testing (Harwood et al., 2004). The availability of genomic sequence data allows for Vibrio spp. comparative genomic studies, revealing not only a wealth of information on their evolution and pathogenesis, but potential targets for molecular typing and detection. Molecular techniques for Vibrio identification and subtyping have been developed, including oligonucleotide probes (Wright et al., 1993) and DNA microarray technologies which target species-specific virulence determinants . PCR-based methods have been d...

Simultaneous differential detection of human pathogenic and nonpathogenicVibriospecies using a multiplex PCR based ongyrB andpntA genes

“…This shows the versatility of PCR test to distinguish V. cholerae from V. mimicus , as both species are genetically similar. Besides V. cholerae and V. mimicus , V. parahaemolyticus is also frequently misinterpreted as V. alginolyticus (Tracz et al. 2007).…”

“…Vibrio species live naturally in aquatic environments and are commonly isolated from marine organisms and seafood. Pathogenic Vibrio species such as Vibrio cholerae , Vibrio parahaemolyticus and Vibrio vulnificus are of major concern as they are responsible for infectious diseases in humans (Tracz et al. 2007).…”

Development of agroELgene-based species-specific multiplex polymerase chain reaction assay for simultaneous detection ofVibrio cholerae,Vibrio parahaemolyticusandVibrio vulnificus

“…; Tracz et al . ). All three species are commonly associated with sea water, sediment, shellfish and the intestinal contents of fish (Wong et al .…”

NontoxigenicVibrio choleraeSepticemia in an Immunocompromised Patient

“…Additionally, against a custom database of 16 reference V. cholerae isolates, this isolate scored 2.507, further substantiating the highly probable species-level identification as V. cholerae . A Vibrio -specific molecular speciation assay [1] was also used to confirm the identity of this isolate. Amplification and DNA sequencing of the ftsZ allele using oligonucleotides ftsZF1 and ftsZR2 for a 1041 bp fragment demonstrated 100% nucleotide identity between this isolate and reference non-O1 V. cholerae strain ATCC 35971, and 99.7% nucleotide identity between this isolate and V. cholerae O1 reference strains ATCC 31503 and NH-V-86.…”