J Med Microbiol 56 (2007), 56-65; DOI: 10.1099/jmm.0.46759-0
© 2007 Society for General Microbiology
ISSN 1473-5644
Rapid detection of Vibrio species using liquid microsphere arrays and real-time PCR targeting the ftsZ locus
Dobryan M. Tracz,
Paul G. Backhouse,
Adam B. Olson,
Joanne K. McCrea,
Julie A. Walsh,
Lai-King Ng and
Matthew W. Gilmour
National Microbiology Laboratory, Canadian Science Centre for Human and Animal Health, Public Health Agency of Canada, 1015 Arlington Street, Winnipeg, Manitoba R3E 3R2, Canada
Correspondence
Matthew W. Gilmour
Matthew_Gilmour{at}phac-aspc.gc.ca
Received 1 June 2006
Accepted 2 October 2006
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.
Abbreviations: Ct, threshold cycle; LUX, Light Upon eXtension; RT-PCR, real-time PCR.
The GenBank/EMBL/DDBJ accession numbers for the ftsZ sequences are DQ520257–DQ520276.
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INTRODUCTION
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Pathogenic 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, 2005, 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 (Panicker et al., 2004). PCR-based methods have been developed to identify and subtype Vibrio spp., including multiplex PCR primer sets for the amplification of tlh/tdh/trh (Bej et al., 1999), ompU/toxR/tcpI/hlyA (Rivera et al., 2001) and vvh/viuB (Panicker et al., 2004). PCR-based molecular-typing studies have been performed in V. cholerae with amplified fragment length polymorphism (AFLP), BOX- and enterobacterial repetitive intergenic consensus (ERIC)-PCR (Singh et al., 2001); in V. parahaemolyticus with multilocus sequence typing (MLST) (Chowdhury et al., 2004); and in V. vulnificus with randomly amplified polymorphic DNA (RAPD) analysis (Parvathi et al., 2005; Warner & Oliver, 1999). Recently, real-time PCR (RT-PCR) protocols have been developed for the identification of V. vulnificus, employing both TaqMAN (Campbell & Wright, 2003; Panicker & Bej, 2005) and SYBR Green technology, the latter targeting the vvhA cytolysin gene alone (Panicker et al., 2004) or in a multiplex real-time assay (Gubala, 2005). Alternatively, genetic variation at core genetic determinants can be exploited for speciation and disease surveillance.
We have previously utilized Light Upon eXtension (LUX) fluorogenic primers and liquid microsphere suspension arrays for the detection and typing of Escherichia coli (Gilmour et al., 2006) and human-adapted Salmonella enterica serovars Typhi and Paratyphi A (Tracz et al., 2006). In contrast to the 5' nuclease system which uses two unlabelled PCR primers and a fluorophore-labelled probe with a quencher, the LUX RT-PCR system requires a single pair of primers, a fluorogenic (that is self-quenching by the formation of a hairpin loop) primer and its unlabelled primer pair (Nazarenko et al., 2002). Luminex microsphere suspension array technology utilizes allele-specific oligonucleotide probes conjugated to fluorescently coded microspheres which capture soluble DNA in a liquid phase and characterize hybridization partners by flow cytometry (Dunbar et al., 2003). In this study, our goal was to apply LUX RT-PCR and Luminex microsphere technology to target species-specific polymorphisms at the ftsZ locus for the molecular typing of Vibrio species.
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METHODS
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Bacterial strains.
Strains of Vibrio spp. (see Table 1
) were obtained from the laboratories listed. Publicly available genomic sequence data were used to initiate comparative genomic studies at the ftsZ locus: V. cholerae O1 (El Tor biovar strain N16961, GenBank accession no. NC_002505); V. parahaemolyticus (RIMD 2210633, NC_004603); and V. vulnificus (CMCP6, NC_004459; YJ016, NC_005139) (Heidelberg et al., 2000; Makino et al., 2003; Chen et al., 2003). Genomic sequence data from other Gram-negative bacteria were also used for assessing overall diversity at ftsZ: E. coli (EDL933, GenBank accession no. NC_002655; K12, NC_000913; CFT073, NC_004431); Shigella flexneri (2457T, NC_004741; 301, NC_004337); Yersinia pestis (Medievalis 91001, NC_005810; KIM, NC_004088; CO92, NC_003143); Salmonella serovar Typhimurium (LT2, NC_003197; DT104, Sanger Institute Microbial Pathogens Group, unpublished data); Salmonella serovar Typhi (CT18, NC_003198; Ty2, NC_004631); Salmonella serovar Paratyphi A (ATCC 9150, NC_006511); Salmonella serovar Enteritidis (LK5, University of Illinois at Urbana-Champaign, unpublished data; www.salmonella.org).
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Table 1. Allelic discrimination of ftsZ in Vibrio spp. using liquid microsphere suspension and RT-PCR assays
Abbreviations: VC, V. cholerae; VP, V. parahaemolyticus; VV, V. vulnificus; +, positive; –, negative; ±, intermediate signal strength.
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Bioinformatics.
Initial screening of polymorphic loci was performed with BLASTN (www.ncbi.nlm.nih.gov/blast/) and multiple sequence alignments were completed using CLUSTALW (www.ebi.ac.uk/clustalw/) and Boxshade (www.ch.embnet.org). Neighbour-joining trees were constructed with Hasegawa–Kishono–Yano (HKY85) distance correction using SplitsTree4 (Huson, 1998), and genetic diversity statistics were calculated with DnaSP 4.10.3 (Rozas et al., 2003), using only segments of ftsZ for which there was data for all examined strains (i.e. regions from complete genome data outside the amplified segments of ftsZ were not included). Split decomposition analysis was performed with SplitsTree4 using alignment inputs created by CLUSTALW and calculated using only parsimony-informative sites.
PCR and sequencing.
Template DNA was prepared by centrifuging a loopful of bacterial culture grown on OMH agar (10 % blood), resuspending the pellet in 1 ml TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and boiling for 10 min. Boiled cell debris was pelleted and the supernatant was removed and used as DNA template in real-time and standard PCR reactions.
Standard PCR was performed with Platinum High Fidelity Taq (Invitrogen), following the manufacturer's directions, with oligonucleotides described in Table 2, on an ABI GeneAmp 9700 thermocycler. PCR conditions were: initial denaturation at 94 °C for 5 min, 35 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for 30 s and extension at 68 °C for 45 s, with a final extension at 68 °C for 5 min. PCR products were purified using the QIAquick PCR purification kit (Qiagen) and sequenced on an ABI 3730. A PCR method for detection of the toxR gene in V. parahaemolyticus was performed on isolate 97-1598 and selected control templates with primers toxRF and toxRR (368 bp PCR product; Table 2) as described elsewhere (Kim et al., 1999).
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Table 2. Oligonucleotide primers and probes used in this study
The species target is indicated in parentheses. Abbreviations: ALEXA 546, Alexa Fluor 546; HEX, 6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein; JOE, 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein; Z, T-phosphorothioate; F, A-phosphorothioate; E, G-phosphorothioate; bases in lower-case type at the 5' end indicate those required for LUX primer hairpin formation and are not present in the target sequence; the penultimate 3' base in lower-case type was labelled with a fluorophore.
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LUX fluorogenic and unlabelled primer pairs (Table 2
) were designed using the D-LUX designer software (Invitrogen) by targeting polymorphic regions in the ftsZ locus. For LUX RT-PCR, Platinum Quantitative PCR SuperMix-UDG (Invitrogen) was used for the amplification mixture with each LUX primer at a final concentration of 200 nM and 2.5 µl template for a total reaction volume of 25 µl. RT-PCR reactions were performed on the SmartCycler 2.0 (Cepheid). Samples were amplified by an initial denaturation at 95 °C for 3 min and 35 cycles of denaturation at 95 °C for 10 s, annealing at 58 °C for 15 s and an extension step at 72 °C for 15 s. Fluorescence was detected at the annealing step and the threshold level was set at 30 fluorescence units. Products for V. cholerae, V. parahaemolyticus and V. vulnificus RT-PCR were 92, 91 and 64 bp, respectively. An RT-PCR reaction was considered positive at the point at which the fluorescent signal exceeded the background level [the cycle threshold (Ct) value], and each strain was tested a minimum of two times.
Liquid microsphere suspension arrays.
Allelic discrimination of ftsZ was achieved after amplifying biotin-labelled target DNA from all Vibrio spp. in a PCR using ftsZ-F1 (common forward primer) and reverse primers ftsZ-R2-L (targeting V. cholerae only; 1215 bp product; Table 2) and ftsZ-R1-L (all other Vibrio species; 
1197 bp product; Table 2). PCR was performed as a 100 µl reaction with Platinum High Fidelity Taq (Invitrogen), following the manufacturer's directions with the following final primer concentrations: ftsZ-F1, 1 µM; ftsZ-R2-L and ftsZ-R1-L, 1.5 µM each. PCR reactions were purified as described above and eluted in 50 µl EB buffer (Qiagen), and T7 exonuclease was used to digest the unlabelled target DNA strand (Gilmour et al., 2006; Nikiforov et al., 1994). DNA digestion was performed by mixing 43 µl purified PCR product with 5 µl buffer 4 (USB) and 2 µl T7 exonuclease (20 U total), and incubating at 37 °C for 1 h. Enzymic digestion was inactivated by adding 2 µl 0.5 M EDTA. Oligonucleotide probes were designed matching the sense strand in regions characteristic of individual allele subtypes and screened using SBEprimer software (Kaderali et al., 2003) for potential secondary structures or cross-hybridization between probes. xMAP carboxylated fluorescently coded microsphere sets 150, 155 and 160 (Luminex) were coupled to oligonucleotides VC-DOB98, VP-DOB99 and VV-DOB100, respectively (Table 2). Hybridization of biotin-labelled ftsZ target DNA strands to the capture probe-coupled microspheres and flow cytometry were performed in triplicate, as previously described (Gilmour et al., 2006). The positive cut-off value for the ftsZ assay was chosen to be five times greater than the value for the negative control.
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RESULTS AND DISCUSSION
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Genetic diversity at the ftsZ locus in the genus Vibrio
Using publicly available genomic sequences deposited in GenBank, including V. cholerae (strain N16961), V. parahaemolyticus (RIMD 2210633) and V. vulnificus (CMCP6, YJ016), we performed a comparative sequence analysis and identified Vibrio species-specific polymorphisms at the ftsZ, trpS and dnaX loci. These genes are core bacterial genetic determinants which serve as useful target loci in predicting overall interspecies genetic relatedness and do not regularly undergo mutation (Zeigler, 2003). Although analysis of the trpS and dnaX loci did not reveal targets that were appropriate for development of molecular assays, multiple sequence alignments of all available ftsZ sequences from the genus Vibrio and other major enteric pathogens suggested that the species-specific polymorphisms could be exploited for the molecular identification of Vibrio species (Fig. 1).
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Fig. 1. Comparative genomic analysis of the ftsZ locus. Phylogeny of (a) Gram-negative organisms and (b) the Vibrio genus based upon a neighbour-joining tree. Accession numbers for sequence data are presented in Methods; strain details are given in parentheses. The scale bar represents the distance score. (c) Multiple sequence alignment of the targeted region of ftsZ from representative isolates from each Vibrio species analysed in this study. LUX primer binding sites are indicated by arrows (with arrowheads representing the 3' end of the primer). Liquid microsphere suspension probes are indicated by lines with a circle. Regions used for oligonucleotide design are indicated with boxes.
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Comparative genetic analysis of ftsZ between Vibrio species and other major Gram-negative enteric pathogens showed that the genus Vibrio has a clear phylogenetic separation at this locus from S. enterica serovars (Typhi, Enteritidis, Typhimurium, Paratyphi A), E. coli, Sh. flexneri and Y. pestis (Fig. 1a). Additionally, the three Vibrio species (V. cholerae, V. parahaemolyticus and V. vulnificus) each encoded distinct ftsZ alleles, and amplification of a >1100 bp section with primers ftsZF1/R2 (V. cholerae) and ftsZF1/R1 (all other Vibrio) allowed for identification of species-specific variation. This genetically diverse region of ftsZ was sequenced for a panel of Vibrio spp., including V. cholerae (strain 93-0302), V. parahaemolyticus (04-1240), V. vulnificus (92-1751), Vibrio alginolyticus (86-148, 97-1598, MWG656), Vibrio mimicus (94-939, 86-852B), Vibrio furnissi (96-0200), Vibrio fluvialis (89-751) and Vibrio metschnikovii (81-66). Comparative analysis of this sequence data and the previously published sequence data demonstrated a close association between V. cholerae and V. mimicus alleles, as well as between V. parahaemolyticus and V. alginolyticus alleles (Fig. 1b). These results corroborate earlier phylogenetic studies on the genus Vibrio, using concatenated gene sequences from 16S rRNA, recA, pyrH and rpoA, which found similar associations between these species (Thompson et al., 2004, 2005).
A total of 19 unique alleles of ftsZ were observed, and the genetic diversity and mutation types were calculated for a 785 bp region for which data were available for all strains. This segment represented 65 % of the complete ftsZ coding sequences (CDS) and encoded a total of 314 polymorphic sites (including 304 synonymous and 10 non-synonymous mutations). As an essential protein in cell division (Margolin, 2005; Errington et al., 2003), adaptive mutation of FtsZ should be rare, and this was reflected in the low ratio of non-synonymous to synonymous mutations (dN/dS=0.03). Split decomposition analysis did not indicate recombination among ftsZ loci (data not shown). In comparison, a segment of the hsp60 locus (556 bp) has previously been sequenced for 14 species (Kwok et al., 2002), and in that dataset we calculated 180 polymorphic sites (of which 167 were synonymous).
RT-PCR identification of V. cholerae, V. parahaemolyticus and V. vulnificus
Although the V. cholerae allele of ftsZ was similar to that of V. mimicus, and the V. parahaemolyticus allele was similar to that of V. alginolyticus, there was sufficient genetic diversity for the design of putatively species-specific primers. Targeting species-specific nucleotide polymorphisms in ftsZ, we developed RT-PCR assays employing LUX fluorogenic primer technology (Fig. 1c). RT-PCR was performed using template from a panel of Vibrio strains from Bangladesh and Canada, including representative type strains and clinical isolates from the major pathogenic species (V. cholerae, V. parahaemolyticus and V. vulnificus), as well as less common human Vibrio pathogens (V. alginolyticus, V. fluvialis, V. furnissi, V. mimicus and V. metschnikovii). The V. cholerae LUX primer set was successful in identifying all V. cholerae templates, with one exception (Table 1, Fig. 2a). All Ct values for the V. cholerae RT-PCR were consistently in the 16 to 19-cycle range (data not shown). V. parahaemolyticus and V. vulnificus LUX RT-PCR correctly identified all V. parahaemolyticus and V. vulnificus templates, respectively (Fig. 2b, c). Notably, there appeared to be two distinct groups of Ct values within the positive V. vulnificus RT-PCR results, one with Ct values of 18–19 and the other with Ct values of 25–28.
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Fig. 2. Allelic discrimination of species-specific polymorphisms in clinically important Vibrio species with ftsZ LUX RT-PCR targeting (a) V. cholerae, (b) V. parahaemolyticus and (c) V. vulnificus. Representative strains tested are indicated in the key; NTC, no template control. The horizontal line represents a threshold of 30 fluorescence units, the cut-off for positive signals (which are indicated with solid lines, whereas negative signals are indicated with dashed lines).
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To confirm results from the LUX RT-PCR assay, we performed additional DNA sequencing of the ftsZ gene in selected Vibrio isolates. Only a single RT-PCR assay yielded an unexpected result, as V. cholerae ATCC 14033 was negative for V. cholerae RT-PCR. Sequencing of V. cholerae ATCC 14033 ftsZ found a 2 nt difference in the forward unlabelled primer sequence, which could prevent efficient primer binding and likely accounts for the negative result in V. cholerae RT-PCR. The two groups of Ct values observed in the V. vulnificus RT-PCR are likely due to the presence of nucleotide polymorphisms in the forward primer-binding region, as the sequenced templates from the late Ct value group had a 2 nt difference in the primer target sequence. Although there was a difference in Ct values among V. vulnificus templates, the V. vulnificus RT-PCR was still effective in differentiating V. vulnificus from other Vibrio spp. Due to the requirements of LUX primer design, no other region in ftsZ offered an alternative target for V. vulnificus RT-PCR with this method. Intra-species allelic diversity has previously been observed for V. cholerae, V. parahaemolyticus and V. vulnificus (Farfan et al., 2002; Chowdhury et al., 2004; Gutacker et al., 2003), therefore it is not surprising that a single allele of ftsZ was not observed for all strains within each species in this study.
Liquid microsphere suspension arrays for ftsZ
Microsphere-based suspension arrays have previously been used for the molecular typing and detection of bacterial pathogens (Borucki et al., 2005; Dunbar et al., 2003; Gilmour et al., 2006; Tracz et al., 2006), and we investigated this technology as an additional technique for the allelic discrimination of ftsZ in Vibrio isolates. Vibrio probes were designed to target a region of ftsZ approximately 50 bp downstream of the LUX RT-PCR target sequences because of a species-specific polymorphism-rich region that could be covered by a single probe of 20 oligonucleotides (Fig. 1c). The VC-DOB98 and VV-DOB100 probes were successful in hybridizing to all V. cholerae and V. vulnificus target DNA, respectively, with one exception (Table 1, Fig. 3). All V. parahaemolyticus isolates had positive hybridizations with the VP-DOB99 probe, which also produced positive or intermediate signals with all V. alginolyticus target DNA templates. Overall, excellent reproducibility of results with the microsphere suspension array was observed between repeat hybridizations on multiple days.
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Fig. 3. Allelic discrimination of species-specific polymorphisms in selected isolates of clinically important Vibrio species using liquid microsphere suspension arrays. SEM is indicated by vertical lines on each bar. The ID of each strain is given in parentheses.
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The Luminex microsphere assay produced several results that required sequence confirmation because of intermediate signals. The VV-DOB100 probe did not hybridize with V. vulnificus 90-208 on three separate occasions, and sequence analysis of the 90-208-encoded ftsZ allele showed an identical probe-binding region to that of other V. vulnificus templates that were positive in the microsphere assay. Additional factors must be influencing the VV-DOB100 probe–template hybridization, and may involve secondary structure or sequence outside the probe-binding region. Positive or intermediate signals were also produced with the VP-DOB99 probe from all V. alginolyticus templates, which were confirmed on multiple hybridization dates. Sequence analysis of these closely related Vibrio spp. revealed a 4–5 nt difference between V. parahaemolyticus and V. alginolyticus in the VP-DOB99 probe region. The presence of these polymorphisms would prevent optimal probe hybridization and likely accounts for the intermediate signal strength in these cases. Overall, positive hybridization results in the Luminex microsphere assay were clearly distinguishable from intermediate non-specific signals, and were useful in differentiating Vibrio spp.
Speciation of isolate 97-1598
Although they are distinct species of the genus Vibrio, it is not unusual for V. parahaemolyticus and V. alginolyticus to be incorrectly identified in the laboratory (Robert-Pillot et al., 2002). Isolate 97-1598 was originally received by our laboratory as a V. parahaemolyticus; however, in the V. parahaemolyticus RT-PCR assay, this isolate was found to be negative, and sequencing of ftsZ determined that this allele was more similar to those encoded by V. alginolyticus. A PCR screen for the toxR gene (Kim et al., 1999) was used to rule out isolate 97-1598 as a V. parahaemolyticus. Isolates 05-1000, 04-1240 and ATCC 17802 encoded the V. parahaemolyticus toxR determinant, whereas 97-1598 and two V. alginolyticus templates (86-148 and MWG656) were negative (data not shown). The results of this PCR test corroborate the results of the LUX primer assay and the sequence analysis for isolate 97-1598. Furthermore, our panel of Vibrio strains initially included an isolate of Vibrio hollisae, which was negative for all LUX RT-PCR and Luminex microsphere assays. However, this strain has been removed from the genus Vibrio and renamed Grimontia hollisae (Thompson et al., 2003).
Advantages and disadvantages of assays targeting ftsZ polymorphisms
In this study, two novel assays were developed for the molecular typing of pathogenic Vibrio. The Luminex liquid microsphere assay involves a PCR that can amplify ftsZ from all clinically relevant Vibrio spp. and utilizes species-specific probes targeting the major Vibrio pathogens. Although the liquid microsphere assay did not have absolute sensitivity and produced several intermediate signals, the results were useful in detection of Vibrio spp. Furthermore, the Luminex platform allows for the future design of additional probes to other Vibrio molecular targets and, in addition to speciation, may offer a powerful multiplex assay for identifying multiple species- and genus-specific virulence targets. The LUX RT-PCR assays were developed as singleplex reactions for each Vibrio sp., but offered many advantages, including speed, reproducibility and ease of primer design. The minimum time required to reach a positive signal in the RT-PCR method was 25 min from the point of template addition. All negative or late Ct signals in RT-PCR were accounted for by sequencing analysis of polymorphisms in the ftsZ primer-binding regions. The unique polymorphisms in ftsZ are excellent targets for molecular typing of clinically important Vibrio spp. and, overall, RT-PCR provided a more specific, rapid and cost-effective method for identifying V. cholerae, V. parahaemolyticus and V. vulnificus.
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ACKNOWLEDGEMENTS
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Thank you to the DNA Core Facility at the National Microbiology Laboratory for DNA sequencing and oligonucleotide synthesis; to Connie Barton (NML) for thoughtful discussions; and to the Food Directorate, Health Canada (Ottowa), the International Centre for Diarrhoeal Disease Research (Bangladesh) and provincial laboratories of British Columbia, Alberta, Saskatchewan, Manitoba, Ontario, Quebec and New Brunswick for use of isolates. We would also like to thank the genome sequencing centres that made their data publicly available, including the Sanger Institute and the University of Illinois at Urbana-Champaign.
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INTRODUCTION
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