Evaluation of Three Extraction Methods for Detection of Erwinia amylovora from Pear Leaves by Real-Time PCR
Samo za registrovane korisnike
2014
Autori
Ivanović, MilanKuzmanović, Nemanja
Prokić, Andjelka
Blagojević, N.
Obradović, Aleksa
Gašić, K.
Članak u časopisu (Objavljena verzija)
Metapodaci
Prikaz svih podataka o dokumentuApstrakt
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.
Ključne reči:
extraction / detection / pear / Erwinia amylovora / real-time PCRIzvor:
Acta Horticulturae, 2014, 1056, 81-84Izdavač:
- International Society for Horticultural Science
Finansiranje / projekti:
- Razvoj integrisanih sistema upravljanja štetnim organizmima u biljnoj proizvodnji sa ciljem prevazilaženja rezistentnosti i unapređenja kvaliteta i bezbednosti hrane (RS-MESTD-Integrated and Interdisciplinary Research (IIR or III)-46008)
DOI: 10.17660/ActaHortic.2014.1056.10
ISSN: 0567-7572
WoS: 000357733400010
Scopus: 2-s2.0-84912063778
Institucija/grupa
Poljoprivredni fakultetTY - 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 . .