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Evaluation of Three Extraction Methods for Detection of Erwinia amylovora from Pear Leaves by Real-Time PCR
dc.creator | Ivanović, Milan | |
dc.creator | Kuzmanović, Nemanja | |
dc.creator | Prokić, Andjelka | |
dc.creator | Blagojević, N. | |
dc.creator | Obradović, Aleksa | |
dc.creator | Gašić, K. | |
dc.date.accessioned | 2020-12-17T21:03:14Z | |
dc.date.available | 2020-12-17T21:03:14Z | |
dc.date.issued | 2014 | |
dc.identifier.issn | 0567-7572 | |
dc.identifier.uri | http://aspace.agrif.bg.ac.rs/handle/123456789/3522 | |
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 | International Society for Horticultural Science | |
dc.relation | info:eu-repo/grantAgreement/MESTD/Integrated and Interdisciplinary Research (IIR or III)/46008/RS// | |
dc.rights | restrictedAccess | |
dc.source | Acta Horticulturae | |
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 | article | |
dc.rights.license | ARR | |
dc.citation.epage | 84 | |
dc.citation.other | 1056: 81-84 | |
dc.citation.spage | 81 | |
dc.citation.volume | 1056 | |
dc.identifier.doi | 10.17660/ActaHortic.2014.1056.10 | |
dc.identifier.scopus | 2-s2.0-84912063778 | |
dc.identifier.wos | 000357733400010 | |
dc.type.version | publishedVersion |