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Evaluation of Three Extraction Methods for Detection of Erwinia amylovora from Pear Leaves by Real-Time PCR
dc.creator | Ivanović, M. | |
dc.creator | Kuzmanović, Nemanja | |
dc.creator | Prokić, Anđelka | |
dc.creator | Blagojević, Nevena | |
dc.creator | Obradović, Aleksa | |
dc.creator | Gašić, Katarina | |
dc.date.accessioned | 2019-04-04T15:59:58Z | |
dc.date.available | 2019-04-04T15:59:58Z | |
dc.date.issued | 2014 | |
dc.identifier.issn | 0567-7572 | |
dc.identifier.uri | https://plantarum.izbis.bg.ac.rs/handle/123456789/315 | |
dc.description.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 extraction 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. | en |
dc.publisher | Int Soc Horticultural Science, Leuven 1 | |
dc.rights | restrictedAccess | |
dc.source | XIII International Workshop on Fire Blight | |
dc.subject | extraction | en |
dc.subject | detection | en |
dc.subject | pear | en |
dc.subject | Erwinia amylovora | en |
dc.subject | real-time PCR | en |
dc.title | Evaluation of Three Extraction Methods for Detection of Erwinia amylovora from Pear Leaves by Real-Time PCR | en |
dc.type | conferenceObject | |
dc.rights.license | ARR | |
dc.citation.epage | 84 | |
dc.citation.other | 1056: 81-84 | |
dc.citation.spage | 81 | |
dc.citation.volume | 1056 | |
dc.type.version | publishedVersion | |
dc.identifier.doi | 10.17660/ActaHortic.2014.1056.10 | |
dc.identifier.scopus | 2-s2.0-84912063778 | |
dc.identifier.wos | 000357733400010 |
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