31 Tan, M. K Ghalayini, A 2008 A 5-plex real-time PCR assay for quantitative detection and identification of Tilletia indica 9th International Congress of Plant Pathology Torino, Italy 90 (S2) 150 Journal of Plant Pathology 24/08/2008 <p>A new molecular test has been developed to help in the surveillance of Karnal bunt, a disease which produces bunted grains with rotting fish smell. Karnal bunt is caused by <em>Tilletia indica</em> and is the subject of strict quarantine regulations by many wheat growing countries including Australia. Any incursion would cause severe disruption to Australia&rsquo;s international wheat trade and consequent huge losses in export markets. The protocol involves the release of DNA from spores, enrichment of DNA from <em>Tilletia </em>species and a final five-plex real-time quantitative PCR assay to detect, identify and distinguish <em>T. indica </em>and other commonly occurring <em>Tilletia </em>species (<em>T. walkeri, T. ehrhartae, T. horrida,</em> and a <em>Tilletia </em>group comprising <em>T. caries, T. trabutii </em>and <em>T. fusca</em>) in wheat grains. This fluorescent molecular tool has a detection sensitivity of 5-10 spores and thus bypasses the germination step in the current quarantine protocol required for confirmation when only a few spores have been found. The single&ndash;tube five-plex realtime assay contains five dual-labelled species-specific probes and associated species-specific primer pairs for the five target <em>Tilletia </em>species. The five different amplification products are quantitated and identified simultaneously by five different fluorescence spectra in a real-time instrument with at least five channels (Rotorgene 6000, Corbett Research, Australia). This protocol with its increased level of detection sensitivity and the technology to multiplex five fluorescent real-time PCR assay in one tube will greatly reduce workload and reagent costs and enables the implementation of an economically sustainable and effective Karnal bunt surveillance program.</p>