Direct real-time PCR: Towards rapid and cost effective application in fire blight diagnosis

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 mu l 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.