Improved fireblight diagnostics using quantitative real‐time PCR detection of <i>Erwinia amylovora </i>chromosomal DNA

Pirc, M.; Ravnikar, Maja; Tomlinson, J. A.; Dreo, T.

doi:10.1111/j.1365-3059.2009.02083.x

Cited by 64 publications

(78 citation statements)

References 37 publications

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“…2 ). The method was transferred from a previously published qPCR assay [ 22 ]. There are also examples in the literature of one step RT-ddPCR assays that target RNA pathogens, such as Rotavirus [ 10 ] and Pepper Mild Mottle virus ( [ 12 ] and Fig.…”

Section: Pcr Amplifi Cationmentioning

confidence: 99%

“…DNA from the pure bacterial cultures and plant extracts was extracted using magnetic bead based QuickPick™ SML Plant DNA kits (Bio-Nobile, Turku, Finland) in a King Fisher™ automated system (Thermo Labsystem) as described previously for E. amylovora [ 22 ], and with a minor modifi cation (440 μl lysate used in the purifi cation).…”

Section: Pcr Amplifi Cationmentioning

confidence: 99%

“…Reaction components are mixed as shown in Table 1 with 4 μl of DNA in a fi nal 20 μl volume. Concentrations of primers and probe are the same as the previously optimized by Pirc et al for the qPCR assay, 900 nM and 250 nM, respectively [ 22 ]. For droplet generation and PCR, the same steps and cycling conditions as the ones described above for a DNA target were followed.…”

Section: Pcr Amplifi Cationmentioning

confidence: 99%

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Droplet Digital PCR for Absolute Quantification of Pathogens

Gutiérrez-Aguirre

Rački

Dreo

et al. 2015

Methods in Molecular Biology

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The recent advent of different digital PCR (dPCR) platforms is enabling the expansion of this technology for research and diagnostic applications worldwide. The main principle of dPCR, as in other PCR-based methods including quantitative PCR (qPCR), is the specific amplification of a nucleic acid target. The distinctive feature of dPCR is the separation of the reaction mixture into thousands to millions of partitions which is followed by a real time or end point detection of the amplification. The distribution of target sequences into partitions is described by the Poisson distribution, thus allowing accurate and absolute quantification of the target from the ratio of positive against all partitions at the end of the reaction. This omits the need to use reference materials with known target concentrations and increases the accuracy of quantification at low target concentrations compared to qPCR. dPCR has also shown higher resilience to inhibitors in a number of different types of samples. In this chapter we describe the droplet digital PCR (ddPCR) workflow for the detection and quantification of pathogens using the droplet digital Bio-Rad platform QX100. We present as an example the quantification of the quarantine plant pathogenic bacterium, Erwinia amylovora.

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Section: Pcr Amplifi Cationmentioning

confidence: 99%

Section: Pcr Amplifi Cationmentioning

confidence: 99%

Section: Pcr Amplifi Cationmentioning

confidence: 99%

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Droplet Digital PCR for Absolute Quantification of Pathogens

Gutiérrez-Aguirre

Rački

Dreo

et al. 2015

Methods in Molecular Biology

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“…qPCR for detection of a quarantine grapevine pathogen, Xylophilus ampelinus [25], was the first qPCR-based technique included in the EPPO diagnostic protocols, and it is still in use today [26]. Other qPCR methods that have been developed at the NIB can detect plant pathogenic viruses (e.g., potato virus Y isolates, Pepino mosaic virus [27]) and bacteria (e.g., Erwinia amylovora), and Flavescence dorée and Bois noir phytoplasma in grapevine [28,29]. Recently, methods using dPCR for detection and quantification of microorganisms have been published [30,31].…”

Section: Microorganisms -Plant Pathogensmentioning

confidence: 99%

Nucleic-acid analysis in new fields of metrology

Camloh

Dreo

Gruden

et al. 2015

17th International Congress of Metrology

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Abstract. Nucleic-acid analysis (NAA) represents a key bioanalysis to which metrological approaches are applied in various fields, such as biotechnology, food safety, public health, and environmental monitoring. NAA enables detection, identification and quantification of nucleic acids of different organisms and matrices. Approaches specific for the target are most common, and real-time polymerase chain reaction (qPCR) analysis is the most frequently used technique. Other methods are being developed that intensively address specific needs. For example, digital PCR enables sensitive detection and accurate absolute quantification without comparison to reference materials, and rapid isothermal approaches provide simple target detection on-site. At the National Institute of Biology, extensive research has been carried out over the last two decades for the development and assessment of NAA techniques and their applications to different fields. Here, we present some of our experiences with the application of metrological approaches to studies of genetically modified organisms and plant pathogenic microorganisms.

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“…syringae. Pirc et al (2007) and Pirc et al (2009) used Real-time PCR to improve E. amylovora diagnostic. Dreo et al (2011) used newly developed and rapid methods (Real-time PCR, serological test -Ea AgriStrip, Bioreb and LAMP) for early detection of the bacterium during the fowering stage and for determination of its concentration.…”

Section: Identificationmentioning

confidence: 99%

The status of Erwinia amylovora in the former Yugoslav Republics over the past two decades

et al. 2013

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Erwinia amylovora, the causal agent of fire blight (FB) on fruit trees and ornamental plants, rapidly spread across eastern Mediterranean countries in the early 1980s. This quarantine bacterium probably arrived in the southern parts of the former Yugoslavia (now FYR Macedonia) from Greece. Based on symptoms, and isolation and identification data, it was concluded that Erwinia amylovora was the causal agent of pear drying in Macedonia (1989). It was the first experimental confirmation of a presence of E. amylovora in the territory of the former Yugoslavia. The presence of E. amylovora was also proved in Serbia that same year. In Bosnia and Herzegovina, FB was detected during 1990. Based on an official report filed with the Federal Ministry of Agriculture in Belgrade, the presence of E. amylovora in Yugoslavia was confirmed (EPPO - Reporting Service, 1991). Therefore, the presence of the bacterium E. amylovora in the territory of Yugoslavia was officially confirmed in 1990. In Croatia, FB was first observed in villages near the border on Serbia in 1995. In Montenegro, FB was first detected in 1996. In Slovenia, FB appeared as late as in 2001. E. amylovora is now present on 10 hosts (pear, wild pear, apple, quince, medlar, mountainash, hawthorn, firethorn, cotoneaster and Japanese quince) in the territory of the former Yugoslav republics. Based on literature data, losses caused by FB in the former Yugoslav republics in the period 1989-1992 were estimated at about 12,000,000 DEM (mostly in Macedonia) and in the period 1992-1996 at 6,000,000 DEM. Total damage in a more recent epiphytotic year in Slovenia (2003) was estimated at about 474,200 EUR. Conventional and up-to-date rapid methods (PCR, ELISA and IF, BIOLOG and API System, FAME and SDS-PAGE) have been used to identify E. amylovora. Mainly preventive measures have been used to control E. amylovora in the former Yugoslav republics. Spraying with copper products has been practiced during the dormant period and in early spring. In rare cases, spraying has been done at the flowering stage (MARYBLYT), but the problem is that no synthetic bactericides are available on the market. When symptoms occur, only mechanical measures are being applied.

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Improved fireblight diagnostics using quantitative real‐time PCR detection of Erwinia amylovora chromosomal DNA

Cited by 64 publications

References 37 publications

Droplet Digital PCR for Absolute Quantification of Pathogens

Droplet Digital PCR for Absolute Quantification of Pathogens

Nucleic-acid analysis in new fields of metrology

The status of Erwinia amylovora in the former Yugoslav Republics over the past two decades

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