TY  - JOUR
AU  - Ivanović, Milan
AU  - Kuzmanović, Nemanja
AU  - Prokić, Andjelka
AU  - Blagojević, N.
AU  - Obradović, Aleksa
AU  - Gašić, K.
PY  - 2014
UR  - http://aspace.agrif.bg.ac.rs/handle/123456789/3522
AB  - 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.
PB  - International Society for Horticultural Science
T2  - Acta Horticulturae
T1  - Evaluation of Three Extraction Methods for Detection of Erwinia amylovora from Pear Leaves by Real-Time PCR
EP  - 84
SP  - 81
VL  - 1056
DO  - 10.17660/ActaHortic.2014.1056.10
ER  - 

@article{
author = "Ivanović, Milan and Kuzmanović, Nemanja and Prokić, Andjelka and Blagojević, N. and Obradović, Aleksa and Gašić, K.",
year = "2014",
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.",
publisher = "International Society for Horticultural Science",
journal = "Acta Horticulturae",
title = "Evaluation of Three Extraction Methods for Detection of Erwinia amylovora from Pear Leaves by Real-Time PCR",
pages = "84-81",
volume = "1056",
doi = "10.17660/ActaHortic.2014.1056.10"
}

Ivanović, M., Kuzmanović, N., Prokić, A., Blagojević, N., Obradović, A.,& Gašić, K.. (2014). Evaluation of Three Extraction Methods for Detection of Erwinia amylovora from Pear Leaves by Real-Time PCR. in Acta Horticulturae
International Society for Horticultural Science., 1056, 81-84.
https://doi.org/10.17660/ActaHortic.2014.1056.10

Ivanović M, Kuzmanović N, Prokić A, Blagojević N, Obradović A, Gašić K. Evaluation of Three Extraction Methods for Detection of Erwinia amylovora from Pear Leaves by Real-Time PCR. in Acta Horticulturae. 2014;1056:81-84.
doi:10.17660/ActaHortic.2014.1056.10 .

Ivanović, Milan, Kuzmanović, Nemanja, Prokić, Andjelka, Blagojević, N., Obradović, Aleksa, Gašić, K., "Evaluation of Three Extraction Methods for Detection of Erwinia amylovora from Pear Leaves by Real-Time PCR" in Acta Horticulturae, 1056 (2014):81-84,
https://doi.org/10.17660/ActaHortic.2014.1056.10 . .