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Evaluation of Three Extraction Methods for Detection of Erwinia amylovora from Pear Leaves by Real-Time PCR

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Authors
Ivanović, M.
Kuzmanović, Nemanja
Prokić, Anđelka
Blagojević, Nevena
Obradović, Aleksa
Gašić, Katarina
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Abstract
In this study, three bacterial DNA extraction procedures were compared prior to real-time PCR. Healthy pear leaves and twigs were crushed in antioxidant maceration buffer and spiked with Erwinia amylovora to final concentrations from 2.1 x 10(6) to 2.1 x 10(1) cells ml(-1). Bacterial DNA was extracted from aliquots of spiked crude extracts using (i) isopropanol, (ii) REDExtract-N-Amp (TM) Plant PCR kit, and (iii) Taylor's modified DNA purification procedure. The ams region of the chromosomal DNA was selected as target for the real-time PCR. In this study, the REDExtract-N-Amp (TM) and Taylor's modified DNA extraction procedure were most successful in removing PCR inhibitors, leading to detection of 2.1x10(2) E. amylovora CFU/ml. At this concentration, pathogen can be efficiently detected in less than 5 h in spite of inhibitors and plant DNA reducing sensitivity of the reaction. These two methods increased amplification efficiency in real-time PCR compared to a simple isopropanol DNA ex...traction procedure from plant tissues, where the lowest detected concentration was 2.1 x 10(4) CFU/ml. In our research, real-time PCR has proven to be very sensitive method for detection of E. amylovora in plant material. It was 100 times more sensitive compared to other conventional PCR procedures.

Keywords:
extraction / detection / pear / Erwinia amylovora / real-time PCR
Source:
XIII International Workshop on Fire Blight, 2014, 1056, 81-84
Publisher:
  • Int Soc Horticultural Science, Leuven 1

DOI: 10.17660/ActaHortic.2014.1056.10

ISSN: 0567-7572

WoS: 000357733400010

Scopus: 2-s2.0-84912063778
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URI
http://plantarum.izbis.bg.ac.rs/handle/123456789/315
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