A real-time multiplex PCR for the identification and typing of Vibrio cholerae

Greig, David R.; Hickey, Tadgh; Boxall, Megan D.; Begum, H; Gentle, Amy; Jenkins, Claire; Chattaway, Marie Anne

doi:10.1016/j.diagmicrobio.2017.11.017

Cited by 10 publications

(7 citation statements)

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“…One colony per sample was inoculated into 490 μl of distilled water and placed in a boiling water bath for 20 minutes to initiate cell lysis. A real‐time PCR was undertaken to determine if the isolates contained any of the six target genes: toxR , ctxA , wbeO1 , wbfO139 , tcpA classical and tcpA El Tor 23. The gene toxR is seen as a marker for V cholerae identification and is found in all isolates of V cholerae , while ctxA gene encodes for cholera toxin, which is usually associated with the pandemic strains O1 and O139.…”

Section: Methodsmentioning

confidence: 99%

Isolation and characterisation of Vibrio cholerae from fish examined postmortem at ZSL London Zoo between 2014 and 2018

et al. 2020

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BackgroundWhen suspect Vibrio cholerae were cultured from fish at ZSL London Zoo, investigations were carried out to determine whether they were possible causes of cholera.MethodsBacterial culture was carried out on fish examined postmortem and colonies were identified using standard techniques including the API 20NE biochemical test kits. Suspect isolates were submitted to the Public Health England laboratory for additional testing. Separately, a number of fish were submitted for routine histopathology.ResultsOn 13 occasions between 2014 and 2018, suspected V cholerae were cultured from individuals of eight different freshwater fish species. Archived cultures for eight of these (from six different fish species) were investigated and seven isolates (from five fish species) were confirmed as V cholerae, but all were non-O1, non-O139 strains. Whole-genome sequencing showed that the five fish species had unique V cholerae multilocus sequence types (three isolates from Aphanius danfordii were identical), all of which were genetically distant from human isolates.ConclusionsThere was no evidence that these isolates could cause cholera. Histopathological changes consistent with vibriosis were seen in several fish, suggesting that V cholerae were causing the disease, but there were also concurrent infections or predisposing stress factors.

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Section: Methodsmentioning

confidence: 99%

Isolation and characterisation of Vibrio cholerae from fish examined postmortem at ZSL London Zoo between 2014 and 2018

et al. 2020

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“…toxR is found in all V. cholerae isolates and is regarded as a marker for species identification. The ctxA encoding cholera toxin is most often, although not exclusively, associated with V. cholerae serotypes O1 and O139 and is a characteristic of these pandemic lineages (23). Variants of tcpA can be used to identify the classical and El Tor biotypes (23).…”

Section: Methodsmentioning

confidence: 99%

“…The ctxA encoding cholera toxin is most often, although not exclusively, associated with V. cholerae serotypes O1 and O139 and is a characteristic of these pandemic lineages (23). Variants of tcpA can be used to identify the classical and El Tor biotypes (23). For in silico predictions, only results that matched to a gene determinant at Ͼ80% nucleotide identity over Ͼ80% target gene length were accepted.…”

Section: Methodsmentioning

confidence: 99%

Evaluation of Whole-Genome Sequencing for Identification and Typing of Vibrio cholerae

et al. 2018

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Epidemiological and microbiological data on strains isolated between April 2004 and March 2018 ( = 836) and held at the Public Health England culture archive were reviewed. The traditional biochemical species identification and serological typing results were compared with the genome-derived species identification and serotype for a subset of isolates ( = 152). Of the 836 isolates, 750 (89.7%) were from a fecal specimen, 206 (24.6%) belonged to serogroup O1, and 7 (0.8%) were serogroup O139; 792 (94.7%) isolates were from patients reporting recent travel abroad, most commonly to India ( = 209) and Pakistan ( = 104). Of the 152 isolates identified by use of kmer, 149 (98.1%) were concordant with those identified using the traditional biochemical approach. Traditional serotyping results were 100% concordant with those of the whole-genome sequencing (WGS) analysis for the identification of serogroups O1 and O139 and classical and El Tor biotypes. was detected in all isolates of O1 El Tor and O139 belonging to sequence type 69 (ST69) and in O1 classical variants belonging to ST73. A phylogeny of isolates belonging to ST69 from U.K. travelers clustered geographically, with isolates from India and Pakistan located on separate branches. Moving forward, WGS data from U.K. travelers will contribute to global surveillance programs and the monitoring of emerging threats to public health and the global dissemination of pathogenic lineages. At the national level, these WGS data will inform the timely reinforcement of direct public health messaging to travelers and mitigate the impact of imported infections and the associated risks to public health.

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“…In this regard, it seems like the detection and quantification of pathogenic Vibrio spp. for marine species should follow the footsteps of its human-pathogen counterparts (such as V. parahaemolyticus [ 39 , 40 ], V. cholerae [ 41 ], or V. vulnificus [ 42 ]) and embark on the molecular era. Despite all of the advantages of qPCR, one major drawback is the fact that this technique is unable to distinguish live and dead bacteria compared to the conventional culturing methods [ 43 ].…”

Section: Introductionmentioning

confidence: 99%

Real-Time PCR Protocol for Detection and Quantification of Three Pathogenic Members of the Vibrionaceae Family

Costa¹,

Ferreira

Simões³

et al. 2022

Microorganisms

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Vibriosis, an often-fatal disease induced by pathogenic members of the Vibrionaceae family, causes severe economic losses in aquacultures. To mitigate/avoid vibriosis outbursts, it is vital to detect and quantify these pathogens as early as possible. However, standard microbiological methods are time-consuming and often underestimate cell counts, which calls for the development of valid alternatives. In this study, real-time polymerase chain reaction (qPCR) was employed to detect the pathogenic species Vibrio alginolyticus, Listonella anguillara, and Vibrio harveyi using a new primer pair targeting the groEL gene. In addition, the DNA extraction efficiency of three methods, two commercial kits and the boiling method, was compared. The most efficient method was the DNeasy Blood and Tissue kit, with a detection limit ranging between 154 and 600 CFU mL−1 in the case of V. alginolyticus and L. anguillara, and 48 CFU mL−1 for V. harveyi. Thus, this study presents the development and evaluation of a method for the early quantification of all three species in saline suspensions. However, the results obtained by spiking a microalgae sample with V. harveyi emphasize the importance of adjusting the DNA control’s standard curve to the relevant extraction matrices, as it affects the DNA extraction efficiency and may hamper an accurate quantification with qPCR.

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A real-time multiplex PCR for the identification and typing of Vibrio cholerae

Cited by 10 publications

References 14 publications

Isolation and characterisation of Vibrio cholerae from fish examined postmortem at ZSL London Zoo between 2014 and 2018

Isolation and characterisation of Vibrio cholerae from fish examined postmortem at ZSL London Zoo between 2014 and 2018

Evaluation of Whole-Genome Sequencing for Identification and Typing of Vibrio cholerae

Real-Time PCR Protocol for Detection and Quantification of Three Pathogenic Members of the Vibrionaceae Family

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