519 Direct Real-Time PCR: Towards Rapid and Cost Effective Application in Fire Blight Diagnosis W.-S. Kim and A.M. Svircev V.O. Stockwell Agriculture and Agri-Food Canada Department of Botany and Plant Pathology 4902 Victoria Ave. North Oregon State University P.O. Box 6000, Vineland Station Corvallis, Oregon 97330 Ontario, L0R 2E0 USA Canada A.J. Castle Department of Biological Sciences Brock University, St. Catharines Ontario, L2S 3A1 Canada Keywords: direct real-time PCR, E. pyrifoliae, E. amylovora, high throughput diagnosis, apple pollen Abstract Current diagnostic tools must be sensitive, cost-effective and afford high throughput. In order to pursue these goals in real-time PCR systems, we developed direct real-time PCR (DRT-PCR) replacing time consuming DNA purification steps with direct pathogen extract buffer (DiPEB). Consequently, it allows pathogen detection from field samples, thus saving costs and processing time. Chromosomal DNA based universal primers/TaqMan probes were designed respectively for specific detection of Erwinia amylovora or Erwinia pyrifoliae. The primers were designed for both SYBR Green and TaqMan systems and detected pathogens regardless of the size, number or lack of plasmids. No cross-amplification between E. amylovora and E. pyrifoliae was observed, allowing duplex DRT-PCR to distinguish one from another simultaneously. Three different extraction methods (sonication, boiling and maceration) were optimized for apple pollen, blossoms and stem samples. The DRT-PCR detection limit was 20 cfu in 25 μl DRT reaction; however, the detection range varied depending on the extraction method and sample type. Potential applications of multiplex DRT-PCR will be useful to understand the dynamics of pathogen populations in the field or perform routine epidemiological studies and vector transmission assays. INTRODUCTION Conventional PCR has contributed to detection of E. amylovora from different sample types with primers driven from plasmid pEA29 (Bereswill et al., 1992) or chromosomal regions (Bereswill et al., 1995). Recently introduced, SYBR Green, TaqMan and Scorpion based quantitative real-time PCR (De Bellis et al., 2007; Salm and Geider, 2004) have enhanced E. amylovora detection. However, due to the cost of real- time PCR and the time-consuming process of DNA extraction, these methods are restricted to small scale diagnosis. Moreover, primers and probes used in these systems were all designed from the sequence of plasmid pEA29 (De Bellis et al., 2007; Salm and Geider, 2004), that limits the detection of strains harbouring different plasmids (McManus and Jones, 1995; Ockey and Thomson, 2004) or no pEA29 (Llop et al., 2006). In this study we developed cost effective and high throughput real-time PCR that does not require previous DNA extraction. Further, specific primers/TaqMan probes for E. amylovora and E. pyrifoliae were developed based on chromosomal DNA sequences, optimizing sensitivity and specificity in a DRT-PCR system in order to provide wide range detection. We also utilized this DRT-PCR system in duplex mode to detect both E. amylovora and Erwinia pyrifoliae (Kim et al., 1999) simultaneously. Proc. XI th IW on Fire Blight Eds.: K.B. Johnson and V.O. Stockwell Acta Hort. 793, ISHS 2008