|Authors: ||M. Pirc, T. Dreo, M. Ravnikar|
|Keywords: ||Erwinia amylovora, enrichment, validation, DNA extraction, Cox|
Fire blight is present in Europe with few areas remaining pathogen free.
In the absence of visual symptoms testing for latent infection gives useful information and can be used in the process of approving places of plant production.
As the number of Erwinia amylovora cells in symptomless samples is expected to be low, their enrichment in liquid media King’s B and CCT is suggested prior to screening tests.
In our study we have transferred the real-time PCR targeting plasmid pEA29 described by Salm and Geider (2004) to ABI 7900 Sequence Detection System apparatus.
Partial validation was performed to check for critical characteristics that might be affected by modifications.
Adapted real-time PCR protocol was applied to 88 symptomless samples and used to assess the suitability of DNA extraction methods.
In analysis of samples during two years, specific real-time PCR signal for E. amylovora was obtained in only 8 samples.
Attempts to isolate bacteria in pure culture were unsuccessful, as expected from low Ct values.
In comparison of different DNA extraction methods with direct analysis of crude enriched extracts in real-time PCR, direct analysis was shown to be at least as good or superior to both DNeasy Plant Mini Kit (Qiagen) for King’s B and simple extraction method for CCT enrichments.
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