Genomics-informed LAMP-PCR design for detection of
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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). 24-Month Meeting Wien (A) – 12-13.6.2012. www.qdetect.org. XAP LAMP assay.

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24-Month Meeting Wien (A) – 12-13.6.2012

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24 month meeting wien a 12 13 6 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)

24-Month Meeting

Wien (A) – 12-13.6.2012

www.qdetect.org


Xap lamp assay

XAP LAMP assay

Xanthomonas arboricola pv. pruni

Bacterial spot of stone fruit (Prunus)

  • Assay designed

  • Validated

  • Ring tested

  • Paper submitted/ accepted

  • EPPO validation?

Andreas Bühlmann – 12-13.6.2012

Q-Detect 24-Month Meeting, Wien, A


24 month meeting wien a 12 13 6 2012

COST873 STSM in MelbourneAustralian isolates of Xap (DPI Melbourne)Talks about LAMP stuff with DPI and AQIS – they seem to be enthusiastic about the method, interested in: Viruses, Bacteria, Insects


Xap ring test

XAP Ring test

ACW Wädenswil, CITA Zaragoza, IVIA Valencia, APR Lisse

Inter- Laboratory concordance

Andreas Bühlmann – 12-13.6.2012

Q-Detect 24-Month Meeting, Wien, A


Xap ring test1

XAP Ring test

ACW Wädenswil, CITA Zaragoza, IVIA Valencia, APR Lisse

Inter- Assay concordance

Cohen J. 1960. A coefficient of agreement for nominal scales. Euc. Psychol. Meas 20 (1): 37-46.

Landis J.R., Koch G.G. 1997. The measurement of observer agreements for categorical data. Biomedics 33: 159-174.

Andreas Bühlmann – 12-13.6.2012

Q-Detect 24-Month Meeting, Wien, A


Xap ring test2

XAP Ring test

ACW Wädenswil, CITA Zaragoza, IVIA Valencia, APR Lisse

Sensitivity and Specificity

SE: Standard error; lo IC 95%: lower limit of the 95% confidence interval; up IC 95%: upper limit of the 95% confidence interval.

Andreas Bühlmann – 12-13.6.2012

Q-Detect 24-Month Meeting, Wien, A


Xap ring test3

XAP Ring test

ACW Wädenswil, CITA Zaragoza, IVIA Valencia, APR Lisse

Talk or poster on next SEF congress (in spanish)

Andreas Bühlmann – 12-13.6.2012

Q-Detect 24-Month Meeting, Wien, A


Ea lamp assay

Ea LAMP assay

Erwinia amylovora

Fire blight of pome fruit (Spiraeoideae)

  • Assay designed

  • Validated

  • Field tested (only CH)

  • Paper about to submit

  • EPPO validation

  • Further sequencing efforts – 96 bacterial strains to further improve knowledge about presence/ absence of diagnostic targets

Andreas Bühlmann – 12-13.6.2012

Q-Detect 24-Month Meeting, Wien, A


24 month meeting wien a 12 13 6 2012

  • Planned/current assays for plant pathogenic bacteria

  • Comparative genomics, primer design, validation with partners on…

  • Pantoea ananatis

  • Pseudomonas syringae pv. actinida

  • Pseudomonas syringae pv. aesculi

  • Xanthomonas oryzae pv. oryzae

  • Xanthomonas oryzae pv. oryzicola

  • Ralstonia solanacearum (necessary?)

  • Suggestions? Xanthomonas fragariae? (Neil and colleagues)

Andreas Bühlmann – 12-13.6.2012

Q-Detect 24-Month Meeting, Wien, A


Import diagnostics

Import diagnostics

  • Swiss quarantine inspection service is planning to buy two 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 only if no mixed samples

    • i.e. single insects

  • Xa 88.2°C

    Ea 86.0°C

    Bt 83.3°C

    Tp 81.8°C

    Andreas Bühlmann – 12-13.6.2012

    Q-Detect 24-Month Meeting, Wien, A


    Streptomycin resistance monitoring with lamp vs taqman

    Streptomycin resistance monitoring with LAMP? vs. TaqMan

    • Point mutations on codon 43 of Ea rpsL leads to streptomycin resistance:

      • AAALysinewild type susceptible

      • AGAArgininestrep resistant

      • ACAThreoninestrep resistant

      • AAYAspariginestrep resistant

    Andreas Bühlmann – 12-13.6.2012

    Q-Detect 24-Month Meeting, Wien, A


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