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30-Month Meeting Zurich – 22.10.2012

Genomics-informed LAMP-PCR design for detection of Xanthomonas arboricola pv. pruni and Erwinia amylovora. Andreas Bühlmann JF Pothier, THM Smits, JE Frey, B Duffy (ACW, Switzerland). 30-Month Meeting Zurich – 22.10.2012. www.qdetect.org. XAP LAMP assay.

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30-Month Meeting Zurich – 22.10.2012

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  1. Genomics-informed LAMP-PCR design for detection of Xanthomonas arboricola pv. pruni and Erwinia amylovora Andreas Bühlmann JF Pothier, THM Smits, JE Frey, B Duffy (ACW, Switzerland) 30-Month Meeting Zurich – 22.10.2012 www.qdetect.org

  2. XAP LAMP assay Xanthomonas arboricola pv. pruni Bacterial spot of stone fruit (Prunus) • Assay designed • Validated • Ring tested • Paper accepted • EPPO validation? 30-Month Meeting Zurich – 22.10.2012

  3. Ea LAMP assay Erwinia amylovora Fire blight of pome fruit (Spiraeoideae) • Assay designed • Validated • Field tested (only CH) • Paper submitted • EPPO validation? • Further sequencing efforts – 96 bacterial strains to further improve knowledge about presence/ absence of diagnostic targets under way….. 30-Month Meeting Zurich – 22.10.2012

  4. Planned/current assays for plant pathogenic bacteria • Comparative genomics, primer design, validation with partners on… • Pantoea ananatis (ready for validation) • Pseudomonas syringae pv. actinida (further genome sequencing under way) • Xanthomonas oryzae pv. oryzae (Valerie Vérdier, F/USA) • Xanthomonas oryzae pv. oryzicola • Suggestions? Xanthomonas fragariae? (Neil and colleagues) 30-Month Meeting Zurich – 22.10.2012

  5. Import diagnostics • Swiss quarantine inspection service is considering Genies… • Would need: • Thrips palmi  • Bactrocera spp.  • Bemisisa tabaci () • Liriomyza trifolii/sativae (>80% of samples) • Multiplexable by melt curve: • Single tube assays make • inspectors lifes easier • Works with single insects Xa 88.2°C Ea 86.0°C Bt 83.3°C Tp 81.8°C 30-Month Meeting Zurich – 22.10.2012

  6. Airport diagnostics CH • LAMP canimproveimport diagnostics • Saves 66% ofsequencingreactions n=251 30-Month Meeting Zurich – 22.10.2012

  7. Streptomycin resistance monitoring with LAMP? vs. TaqMan • Point mutations on codon 43 of Ea rpsL leads to streptomycin resistance: • AAA Lysine wild type susceptible • AGA Arginine strep resistant • ACA Threonine strep resistant • AAY Asparigine strep resistant 30-Month Meeting Zurich – 22.10.2012

  8. Streptomycin resistance monitoring with LAMP? vs. TaqMan • LAMP is not specific enough to detect SNPs • Same as TaqMan • LNA does not increase specificity but Speed. 30-Month Meeting Zurich – 22.10.2012

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