Development and preliminary evaluation of a real-time PCR assay for Halioticida noduliformans in abalone tissues

Abstract

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.