Real‐time PCR for detection and quantification of <i>Erwinia amylovora</i>, the causal agent of fireblight

Salm, Heike; Geider, Klaus

doi:10.1111/j.1365-3059.2004.01066.x

Cited by 53 publications

(60 citation statements)

References 32 publications

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“…However, these commonly used molecular methods for quantification have several limitations. Classical PCR assays are not quantitative and need separation of the products on agarose gels and visualisation under UV light (Salm and Geider 2004). DNA hybridisation methods are time-consuming and labour-intensive.…”

Section: Introductionmentioning

confidence: 99%

Quantification of viable Candidatus Liberibacter asiaticus in hosts using quantitative PCR with the aid of ethidium monoazide (EMA)

et al. 2009

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Citrus Huanglongbing (HLB) is a devastating disease of citrus known to be associated with a fastidious, phloem-limited Gram-negative, yet to be cultured bacterium in the genus Candidatus Liberibacter. In the present study we have developed a method to quantify viable Candidatus Liberibacter asiaticus (Las) with the aid of ethidium monoazide (EMA) which can differentiate live from dead cells. First, calibration curves were developed with the aid of quantitative real-time PCR (QPCR) by using a plasmid template consisting of a 703 bp DNA fragment of rplKAJL-rpoBC (β-operon) region. Standard equations were then developed to quantify Las genome equivalents in citrus, periwinkle, and Asian citrus psyllid, Diaphorina citri. To overcome the limitation of quantitative PCR in discriminating between live and dead bacterial cells, EMA was used to inhibit the amplification of DNA from the dead cells of Las in plant samples. By using the standard equations and EMA-QPCR methods developed in this study, we found that the proportion of viable cells in citrus and periwinkle ranged from 17-31% and 16-28%, respectively. It was determined that a minimum bacterial concentration is required for HLB symptom development by quantifying the population of Las in symptomatic and asymptomatic leaves. The EMA-QPCR methodology developed in the present study should provide an accurate assessment of viable HLB pathogen, providing a tool to investigate disease epidemiology and thus act as a crucial component for disease assessment and management.

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Section: Introductionmentioning

confidence: 99%

Quantification of viable Candidatus Liberibacter asiaticus in hosts using quantitative PCR with the aid of ethidium monoazide (EMA)

et al. 2009

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“…Plasmid pEA29 is present in vast majority of E. amylovora strains and seems to be specific to E. amylovora, while cross-reactions and lower sensitivity were sometimes reported in PCRs targeting chromosomal targets (Bereswill et al 1995;Maes et al 1996;Llop et al 2000;Roselló et al 2002). Not surprisingly therefore, the first real-time PCR developed for E. amylovora was designed to detect a target sequence on the pEA29 plasmid (Salm and Geider 2004). Similarly, De Bellis et al (2007) have chosen pEA29 as a target in their proof-of-principle paper combining Scorpion primers with nested PCR.…”

Section: Real-time Pcr Assays For Detection and Identification Of Erwmentioning

confidence: 99%

“…First real-time PCR for detection of E. amylovora (Salm and Geider 2004) included SYBR Green and TaqMan chemistry (hydrolysis probes). SYBR Green, an intercalating dye, is included in the reaction mixture and specifically binds to double stranded DNA and emits fluorescence upon binding.…”

Section: Real-time Pcr Assays For Detection and Identification Of Erwmentioning

confidence: 99%

“…Since its publication, new methods became available or were further developed. In addition to many PCR methods, several real-time PCR assays are now available (Salm and Geider 2004;De Bellis et al 2007;Lehman et al 2008;Mohammadi et al 2009;Pirc et al 2009;Svircev et al 2009), previously reviewed by Dreo (2009). In this review, we complete the data on published real-time PCR assay (including Gottsberger 2010), summarize the use and potential of real-time PCR methods in the detection of fire blight and describe transfer and optimization of a previously developed Ams assay (Pirc et al 2009) onto a faster SmartCycler Ò real-time PCR detection system.…”

Section: Introduction: Fire Blight and Real-time Pcrmentioning

confidence: 99%

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Real-time PCR, a method fit for detection and quantification of Erwinia amylovora

Dreo

Pirc

Ravnikar

2012

Trees

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Fire blight, a devastating disease of pome fruit trees continues to pose threat to agricultural production. Detection of its causative agent, bacterium Erwinia amylovora, is usually straightforward in symptomatic samples. Methods with increased sensitivity however, are sometimes needed for detection of E. amylovora and real-time PCR assays have been shown to have required sensitivity and reliability. Here we summarize our previous results on realtime PCR detection of fire blight and present new, fast and sensitive real-time PCR assay based on amsC gene performed on SmartCycler Ò instrument. The setting is optimal for analysis of small number of samples in the laboratory or for on-site detection. Many advantages of real-time PCR assays warrant their use in detection and diagnosis of E. amylovora, particularly in detection of low concentrations of target bacteria e.g. in testing for latent infections. It is to be expected that the use of real-time PCR will increase in both diagnostics and in research, as a tool for target detection and quantification as well as for gene expression analysis.

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“…For immunological detection and identification purposes there are different methods, but the most often recommended is the double-antibody sandwich indirect enzyme-linked immunosorbent assay (DASI-ELISA) method, based on monoclonal antibodies (Gorris et al 1996). Nucleic acid-based methods can be used for conventional or real-time PCR, with specific primers targeted to the chromosome or to the plasmids (Bereswill et al, 1992(Bereswill et al, , 1995McManus and Jones, 1995;Maes et al, 1996;Llop et al, 2000;Salm and Geider, 2004). However, some strains lack plasmids and may give false-negative reactions with plasmid-based assays (Llop et al 2006).…”

Section: Detection and Identification Of The Pestmentioning

confidence: 99%

Scientific Opinion on the pest categorisation of Erwinia amylovora (Burr.) Winsl. et al.

Rossi¹

2014

EFS2

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The European Commission requested the EFSA Panel on Plant Health to perform the pest categorisation for Erwinia amylovora, which is the causal agent of fire blight. E. amylovora is a plant pathogenic bacterium regulated by the Directive 2000/29/EC (Annexes II-A-II). E. amylovora is a single taxonomic entity. This organism can be accurately identified, based on a range of discriminative methods. Detection methods are available for symptomatic and asymptomatic plant material. E. amylovora is present in all EU Member States except Estonia, Finland and Malta, where host plants are not widely distributed or are rare. The host plants (mainly pear and apple) are cultivated throughout Europe where environmental conditions are conducive to disease development. Although no recent data are available on losses caused by E. amylovora in the EU, fire blight is considered to be the most destructive disease on pear and apple owing to the loss of trees. The analysis of past disease outbreaks previously reported in the EU highlights their considerable potential to have a severe impact on commercial horticulture, especially on apple, pear and quince, as well as on ornamentals and on nursery trade. The disease causes a range of symptoms on the aerial parts of plants, including the fruits, and E. amylovora often kills the trees and causes destructive outbreaks. Contaminated rootstocks, cuttings and grafted trees for transplanting, beehive transportation, rain and wind, are responsible for medium-and longdistance dissemination of the pathogen. Existing control is mainly based on prevention and exclusion. The use of chemical or biological products can prevent infection, and sanitation methods applied to infected plants can control the disease to a certain extent. No curative chemical control agents are available that eradicate E. amylovora in infected orchards.

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Real‐time PCR for detection and quantification of Erwinia amylovora, the causal agent of fireblight

Cited by 53 publications

References 32 publications

Quantification of viable Candidatus Liberibacter asiaticus in hosts using quantitative PCR with the aid of ethidium monoazide (EMA)

Quantification of viable Candidatus Liberibacter asiaticus in hosts using quantitative PCR with the aid of ethidium monoazide (EMA)

Real-time PCR, a method fit for detection and quantification of Erwinia amylovora

Scientific Opinion on the pest categorisation of Erwinia amylovora (Burr.) Winsl. et al.

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