Evaluation of Three Extraction Methods for Detection of Erwinia amylovora from Pear Leaves by Real-Time PCR
Нема приказа
Аутори
Ivanović, M.Kuzmanović, Nemanja
Prokić, Anđelka
Blagojević, Nevena
Obradović, Aleksa
Gašić, Katarina
Конференцијски прилог (Објављена верзија)
Метаподаци
Приказ свих података о документуАпстракт
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.
Кључне речи:
extraction / detection / pear / Erwinia amylovora / real-time PCRИзвор:
XIII International Workshop on Fire Blight, 2014, 1056, 81-84Издавач:
- Int Soc Horticultural Science, Leuven 1
DOI: 10.17660/ActaHortic.2014.1056.10
ISSN: 0567-7572
WoS: 000357733400010
Scopus: 2-s2.0-84912063778
Институција/група
IZBISTY - CONF AU - Ivanović, M. AU - Kuzmanović, Nemanja AU - Prokić, Anđelka AU - Blagojević, Nevena AU - Obradović, Aleksa AU - Gašić, Katarina PY - 2014 UR - https://plantarum.izbis.bg.ac.rs/handle/123456789/315 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 - Int Soc Horticultural Science, Leuven 1 C3 - XIII International Workshop on Fire Blight 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 -
@conference{ author = "Ivanović, M. and Kuzmanović, Nemanja and Prokić, Anđelka and Blagojević, Nevena and Obradović, Aleksa and Gašić, Katarina", 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 = "Int Soc Horticultural Science, Leuven 1", journal = "XIII International Workshop on Fire Blight", 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 XIII International Workshop on Fire Blight Int Soc Horticultural Science, Leuven 1., 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 XIII International Workshop on Fire Blight. 2014;1056:81-84. doi:10.17660/ActaHortic.2014.1056.10 .
Ivanović, M., Kuzmanović, Nemanja, Prokić, Anđelka, Blagojević, Nevena, Obradović, Aleksa, Gašić, Katarina, "Evaluation of Three Extraction Methods for Detection of Erwinia amylovora from Pear Leaves by Real-Time PCR" in XIII International Workshop on Fire Blight, 1056 (2014):81-84, https://doi.org/10.17660/ActaHortic.2014.1056.10 . .