Development and preliminary evaluation of a real-time PCR assay for Halioticida noduliformans in abalone tissues
Diseases of Aquatic Organisms
Biodiversity and Conservation Biology, University of Western Cape, Private Bag X17, Bellville, South Africa; Department of Agriculture, Forestry and Fisheries, Aquaculture Research, Private Bag X2, Roggebaai, Cape Town, 8012, South Africa
Abalone Haliotis midae exhibiting typical clinical signs of tubercle mycosis were discovered in South African culture facilities in 2006, posing a significant threat to the industry. The fungus responsible for the outbreak was identified as a Peronosporomycete, Halioticida noduliformans. Currently, histopathology and gross observation are used to diagnose this disease, but these 2 methods are neither rapid nor sensitive enough to provide accurate and reliable diagnosis. Realtime quantitative PCR (qPCR) is a rapid and reliable method for the detection and quantification of a variety of pathogens, so therefore we aimed to develop a qPCR assay for species-specific detection and quantification of H. noduliformans. Effective extraction of H. noduliformans geno - mic DNA from laboratory grown cultures, as well as from spiked abalone tissues, was accomplished by grinding samples using a pellet pestle followed by heat lysis in the presence of Chelax- 100 beads. A set of oligonucleotide primers was designed to specifically amplify H. noduliformans DNA in the large subunit (LSU) rRNA gene, and tested for cross-reactivity to DNA extracted from related and non-related fungi isolated from seaweeds, crustaceans and healthy abalone; no crossamplification was detected. When performing PCR assays in an abalone tissue matrix, an environment designed to be a non-sterile simulation of environmental conditions, no amplification occurred in the negative controls. The qPCR assay sensitivity was determined to be approximately 0.28 pg of fungal DNA (∼2.3 spores) in a 25 μl reaction volume. Our qPCR technique will be useful for monitoring and quantifying H. noduliformans for the surveillance and management of abalone tubercle mycosis in South Africa. © Inter-Research 2012.
bioassay; environmental conditions; fungal disease; histopathology; host-pathogen interaction; matrix; mitochondrial DNA; monitoring; polymerase chain reaction; population outbreak; seaweed; South Africa; Bacteria (microorganisms); Crustacea; Fungi; Haliotidae; Haliotis midae; Lonchocarpus glaucifolius; fungal DNA; animal; article; classification; fungus; genetics; isolation and purification; methodology; microbiology; mollusc; real time polymerase chain reaction; sensitivity and specificity; species difference; Animals; DNA, Fungal; Fungi; Mollusca; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity; Species Specificity