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PWN detection by LAMP on trapped sawyer beetles ( Monochamus galloprovincialis )

PWN detection by LAMP on trapped sawyer beetles ( Monochamus galloprovincialis ). Mauro Simonato, Jenny Tomlinson, Andrea Battisti, Neil Boonham. LAMP assay design for the pine wood nematode.

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PWN detection by LAMP on trapped sawyer beetles ( Monochamus galloprovincialis )

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  1. PWN detection by LAMP on trapped sawyer beetles (Monochamusgalloprovincialis) Mauro Simonato, Jenny Tomlinson, Andrea Battisti, Neil Boonham

  2. LAMP assay design for the pine wood nematode • LAMP assay design on ITS region of B. xylophilusfollowing a modified protocol by Kikuchi et al. 2009 • Test on DNA extract of B.xylophilus for: • specificity • sensibility Wien 2012

  3. Test of specificity B. xylophilus (positive control) Negative controls Wien 2012

  4. Test of sensibility B. xylophylus Serial dilutions 10-1 10-2 10-3 10-4 10-5 Annealing Temperature for B. xylophilus (ITS): 89.9 °C Wien 2012

  5. Test of sensibility TaqMan Real Time PCR (satellite region): same sensitivity as LAMP 10-1 B. xylophilus 10-2 10-3 10-4 10-5 Wien 2012

  6. Test for cross reactions B.xylophilus DNA dilution + M. galloprovincialis DNA 10-1 10-2 10-3 10-4 10-5 Wien 2012

  7. LAMP assay design for the vector insect • LAMP assay design on COI gene of Monochamus galloprovincialis (positive control for LAMP assay on PWN) • Genes available on GenBank: COI and 16S • Not suitable regions for specific primers of M. galloprovincialis or Monochamus sp.: primer set designed on COI, specific for long horn beetles (Cerambycidae) Wien 2012

  8. LAMP test on long horn beetles Cerambycidae M. galloprovincialis M. galloprovincialis M. sutor Acantocinus aedilis M. sutor Spondylis buprestoides Rhagium inquisitor Arophalus rusticus M. galloprovincialis T annealing (M. galloproncialis): 81.6 °C Other Coleoptera Wien 2012

  9. Multiplex M. galloprovincialis/ B. xylophilus Primer B.x./M.g. Only when B.x.DNA not present 1:1 M. galloprovincialis B.xylophilus 1:2 Wien 2012

  10. Testing LAMP in the PWN monitoring • Screening for PWN on insects sampled in the field: • Sampling in natural forest and harbours with dried multifunnel traps between May-September 2011 • species collected: Monochamus galloprovincialis, and other long horn beetles Wien 2012

  11. Screening for PWN on samples of M. galloprovincialisthrough LAMP • 46 samples analyzed (35 M. galloprovincialis; 11 other long horn beetles) • Half insect thorax used for the DNA extraction • Extraction with Salting Out protocol • LAMP: 2 runs per sample Wien 2012

  12. Results • Only 1 on 46 samples resulted positive but not confirmed by a second run • Some samples showed a amplification product later than the last positive result obtained in the sensibility test Positive control Wien 2012

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