Abstract
DNA- and RNA-based PCR and reverse-transcription real-time PCR assays were developed for diagnostic detection of the vcpA zinc-metalloprotease implicated in the virulence of the coral pathogen Vibrio coralliilyticus. Both PCR methods were highly specific for V. coralliilyticus and failed to amplify strains of closely-related Vibrio species. The assays correctly detected all globally occurring V. coralliilyticus isolates including a newly-described isolate [TAV24] infecting gorgonians in the Mediterranean Sea and highlighted those isolates that had been potentially misidentified, in particular V. tubiashii strains ATCC 19105 and RE22, historically described as important oyster pathogens. The real-time assay is sensitive, detecting 10 gene copies and the relationships between gene copy number and cycle threshold (CT) were highly linear (R2≥99.7). The real-time assay was also not affected by interference from non-target DNA. These assays are useful for rapid detection of V. coralliilyticus and monitoring of virulence levels in environmental samples, allowing for implementation of timely management steps to limit and possibly prevent losses due to V. coralliilyticus infection, as well as furthering investigations of factors affecting pathogenesis of this important marine pathogen.
Highlights
Coral reefs represent one of the most biologically diverse ecosystems in the world, as well as playing a vital role in supporting local communities by way of coastal protection, food production and tourism [1,2,3]
The relationship of the metalloproteases to pathogenicity amongst the prokaryotes is well accepted [22] and their efficacy as virulence factors has been studied in a number of Vibrio species including V. tubiashii [37,53], V. splendidus [54,55], V. vulnificus [56,57,58,59] and V. anguillarum [60]
This study successfully developed specific primers to detect the vcpA gene in V. coralliilyticus using both PCR and reverse transcription real-time PCR approaches
Summary
Coral reefs represent one of the most biologically diverse ecosystems in the world, as well as playing a vital role in supporting local communities by way of coastal protection, food production and tourism [1,2,3]. V. coralliilyticus is amongst the best studied [5,8,9,10,11,12,13,14,15,16,17,18,19,20,21] and investigations into strains isolated from white syndrome disease outbreaks in the Indo-Pacific [6] revealed the expression of a zinc-metalloprotease demonstrated to cause coral tissue damage [15]. The zincmetalloprotease of V. coralliilyticus has been shown to cause coral tissue lesions [8,15] concurrent with white syndrome aetiology and so detection of this important virulence factor could provide early indications of infection and assist agencies in developing strategies to effectively contain coral disease outbreaks. We describe the development of qualititative PCR and quantitative reverse transcription real-time PCR (using SYBR Green I dye) assays with an improved capacity for strain classification and for quantification of viable and active V. coralliilyticus populations
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