Inter-laboratory evaluation of a duplex RT-PCR method using crude extracts for the simultaneous detection of Prune dwarf virus and Prunus necrotic ringspot virus

Massart, Sébastien; Brostaux, Yves; Barbarossa, L.; Cesar, V.; Cieślińska, M.; Dutrecq, O.; Fonseca, Filomena; Guillem, Rhian; Laviña, A.; Olmos, Antonio; Steyer, Stéphan; Wetzel, Thierry; Kummert, J.; Jijakli, M. Haïssam

doi:10.1007/s10658-008-9322-1

Cited by 28 publications

(22 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As a next step, the adoption of a methodology as a new diagnostic test must rely on its inter-laboratory evaluation (Massart et al 2008). Therefore, before any application in routine setting, an inter-laboratory evaluation of this protocol should be carried out to evaluate its robustness and performances in various certification laboratories.…”

Section: Discussionmentioning

confidence: 99%

Development of the simultaneous detection of Ralstonia solanacearum race 3 and Clavibacter michiganensis subsp. sepedonicus in potato tubers by a multiplex real-time PCR assay

2013

View full text Add to dashboard Cite

Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum (Smith) Yabuuchi et al. race 3 are the causal agents of ring-rot and brown-rot of potato respectively. These diseases represent a serious threat to potato production in temperate climates. Both bacteria are listed as A2 pests in the EPPO region and as zero-tolerance quarantine organisms in the European Union. All the detection tests developed so far were only focused on the detection of a single pathogen while the absence of both bacteria has to be certified in the seed tubers. We have therefore developed a new multiplex real-time PCR assay to simultaneously detect both bacteria in a single assay. Additionally, the reliability of this molecular diagnostic test has been improved by the simultaneous amplification of an internal control, corresponding to a potato gene co-extracted from the sample. The polyvalence and the specificity of each set of bacterial primers and probes were evaluated on more than 90 bacterial strains. The limit of detection of this triplex real-time protocol was similar to those observed with other molecular protocols previously developed for the individual detection of one of these bacteria. A concordance of 100 % was obtained in a blind test mimicking the routine application of the technology. In conclusion, this new protocol represents a straightforward and convenient method potentially adapted to primary screening of potato tubers.

show abstract

Section: Discussionmentioning

confidence: 99%

Development of the simultaneous detection of Ralstonia solanacearum race 3 and Clavibacter michiganensis subsp. sepedonicus in potato tubers by a multiplex real-time PCR assay

2013

View full text Add to dashboard Cite

show abstract

“…PDV-infected trees often remain symptomless but necrotic and chlorotic spots on cherry leaves have also been reported (Massart et al, 2008). Sweet cherry trees infected by PNRSV exhibit an array of symptoms ranging from none to a severe rugose mosaic disease, depending upon the virus strain or isolate (Howell and Mink, 1988).…”

Section: Introductionmentioning

confidence: 99%

Main viruses in sweet cherry plantations of Central-Western Spain

Sánchez

Morales-Corts

Benavides

et al. 2015

Sci. agric. (Piracicaba, Braz.)

View full text Add to dashboard Cite

“…In the present study, an extraction buffer newly developed by the Plant Pathology Department of the Gembloux Agriculture University and used previously for detection of plant viruses (Busogoro et al, 2008;Massart et al, 2008) was tested for 'Ca. P. mali' detection.…”

Section: Discussionmentioning

confidence: 99%

A simple and rapid protocol of crude DNA extraction from apple trees for PCR and real-time PCR detection of ‘Candidatus Phytoplasma mali’

Aldaghi

Massart

Dutrecq

et al. 2009

Journal of Virological Methods

Self Cite

View full text Add to dashboard Cite

a b s t r a c tDifferent PCR protocols have been established for detection of European fruit trees phytoplasmas; however the majority of the procedures for extracting phytoplasma DNA are complex, time consuming, and expensive, with a risk of contamination or loss of target DNA. In present study, a crude extract preparation method previously used to detect other plant pathogens was adapted to samples from apple trees infected by 'Candidatus Phytoplasma mali'. End-point and real-time PCR detection of 'Ca. P. mali' were used to compare this extraction procedure with an established method for efficient extraction of purified DNA. The crude extract proved fully adequate for phytoplasma detection in samples from 86 in vitro and 35 in vivo apple shoots or plants and 10 periwinkle plants. High inter-and intra-run reproducibility was obtained for phytoplasma detection with different TaqMan MGB-or SYBR Green-based real-time PCR protocols applied to the crude extracts. Real-time PCR applied to serially diluted crude and purified extracts revealed the same phytoplasma detection limit (dilution up to 10 5 ). All results confirm the suitability of this simple, quick, efficient extraction technique for accurate detection of 'Ca. P. mali' in different types of apple and periwinkle samples.

show abstract

Inter-laboratory evaluation of a duplex RT-PCR method using crude extracts for the simultaneous detection of Prune dwarf virus and Prunus necrotic ringspot virus

Cited by 28 publications

References 29 publications

Development of the simultaneous detection of Ralstonia solanacearum race 3 and Clavibacter michiganensis subsp. sepedonicus in potato tubers by a multiplex real-time PCR assay

Development of the simultaneous detection of Ralstonia solanacearum race 3 and Clavibacter michiganensis subsp. sepedonicus in potato tubers by a multiplex real-time PCR assay

Main viruses in sweet cherry plantations of Central-Western Spain

A simple and rapid protocol of crude DNA extraction from apple trees for PCR and real-time PCR detection of ‘Candidatus Phytoplasma mali’

Contact Info

Product

Resources

About