Detection of Potato Leafroll Virus in Dormant Potato Tubers by Immunocapture and a Fluorogenic 5 prime Nuclease RT-PCR Assay

Schoen, C.D.; Knorr, David A.; Leone, G.

doi:10.1094/phyto-86-993

Cited by 70 publications

(39 citation statements)

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“…In plant pathology, reviews of interest have focused on crop biosecurity (Schaad et al, 2003) and on the detection of seed-borne fungal pathogens (Taylor et al, 2001a) and phytopathogenic bacteria (Schaad and Frederick, 2002). However, realtime PCR detection systems are available for viruses (Schoen et al, 1996;Eun et al, 2000;Finetti Sialer et al, 2000a, b;Roberts et al, 2000;Boonham et al, 2002;Korimbocus et al, 2002), nematodes (Ciancio et al, 2000;Bates et al, 2002), bacteria (Weller et al, 2000;Hermansson and Lindgren, 2001) and fungi (Table 1).…”

Section: Introductionmentioning

confidence: 99%

Real-time quantitative PCR: a new technology to detect and study phytopathogenic and antagonistic fungi

Schena

Nigro

Ippolito

et al. 2004

European Journal of Plant Pathology

282

172

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Real-time PCR technologies open increasing opportunities to detect and study phytopathogenic and antagonistic fungi. They combine the sensitivity of conventional PCR with the generation of a specific fluorescent signal providing real-time analysis of the reaction kinetics and allowing quantification of specific DNA targets. Four main chemistries are currently used for the application of this technique in plant pathology. These chemistries can be grouped into amplicon sequence non-specific (SYBR Green I) and sequence specific (TaqMan, Molecular beacons, and Scorpion-PCR) methods. Amplicon sequence nonspecific methods are based on the use of a dye that emits fluorescent light when intercalated into doublestranded DNA. Amplicon sequence specific methods are based on the use of oligonucleotide probes labelled with a donor fluorophore and an acceptor dye (quencher). The fluorescent signal eliminates the requirement for post-amplification processing steps, such as gel electrophoresis and ethidium bromide staining. This significantly reduces time and labour required for the analysis and greatly increases the throughput of PCR testing as an automated diagnostic system, making it suitable for large-scale analyses. Furthermore, the use of different fluorescent dyes facilitates the detection of several target microrganisms in a single reaction (multiplex-PCR). Real-time PCR makes possible an accurate, reliable and high throughput quantification of target fungal DNA in various environmental samples, including hosts tissues, soil, water and air, thus opening new research opportunities for the study of diagnosis, inoculum threshold levels, epidemiology and host-pathogen interactions. Moreover, the quantification of specific mRNA transcription by real-time PCR is being increasingly applied to the study of changes in gene expression in response to phytopathogenic and antagonistic fungi.

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Section: Introductionmentioning

confidence: 99%

Real-time quantitative PCR: a new technology to detect and study phytopathogenic and antagonistic fungi

Schena

Nigro

Ippolito

et al. 2004

European Journal of Plant Pathology

282

172

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“…Fluorogenic PCR-based (TaqMan) assays have recently shown promise in the detection of a variety of organisms, including clinical bacteria (4,5,19) and plant pathogenic potato leaf roll virus (23). In addition, a TaqMan assay for detection of the potato ring rot bacterium, Clavibacter michiganensis subsp.…”

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confidence: 99%

Detection of Ralstonia solanacearum Strains with a Quantitative, Multiplex, Real-Time, Fluorogenic PCR (TaqMan) Assay

Weller¹,

Elphinstone²,

Smith³

et al. 2000

Appl Environ Microbiol

441

354

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A fluorogenic (TaqMan) PCR assay was developed to detect Ralstonia solanacearum strains. Two fluorogenic probes were utilized in a multiplex reaction; one broad-range probe (RS) detected all biovars of R. solanacearum, and a second more specific probe (B2) detected only biovar 2A. Amplification of the target was measured by the 5 nuclease activity of Taq DNA polymerase on each probe, resulting in emission of fluorescence. TaqMan PCR was performed with DNA extracted from 42 R. solanacearum and genetically or serologically related strains to demonstrate the specificity of the assay. In pure cultures, detection of R. solanacearum to >10 2 cells ml ؊1 was achieved. Sensitivity decreased when TaqMan PCR was performed with inoculated potato tissue extracts, prepared by currently recommended extraction procedures. A third fluorogenic probe (COX), designed with the potato cytochrome oxidase gene sequence, was also developed for use as an internal PCR control and was shown to detect potato DNA in an RS-COX multiplex TaqMan PCR with infected potato tissue. The specificity and sensitivity of the assay, combined with high speed, robustness, reliability, and the possibility of automating the technique, offer potential advantages in routine indexing of potato tubers and other plant material for the presence of R. solanacearum.Ralstonia solanacearum (Smith) (30) is the agent of bacterial wilt, infecting over 450 plant species, including many economically important crops (12). This species has been subclassified into biovars based on biochemical tests and host-dependent races. Biovar 2A (equivalent to race 3) is adapted to temperate climates, has a narrow host range, and is responsible for recent outbreaks of potato brown rot disease in several countries of Western Europe and elsewhere worldwide (13,27). Although other biovars can also infect potatoes, biovar 2A is the most destructive phenotype in temperate areas.R. solanacearum is listed as a quarantine organism in the European Union (EU) (2), where new legislation has been introduced to control and eradicate the organism (3). Latent infections in seed potato tubers (6) have lead to the spread of the organism, both locally and internationally, and effective control of brown rot is dependent on the reliability of detection of the pathogen at this latent stage. For practical purposes, a detection assay is required which is rapid, specific, and sensitive to levels lower than those occurring in naturally infected potatoes and should be applicable to a crude sample of the specimen of interest (25). Serological techniques such as immunofluorescence (IF) microscopy, the enzyme-linked immunosorbent assay (ELISA) (10,15,21), and molecular techniques involving the PCR (9, 24) have been described for detection of R. solanacearum. An EU control directive (3) allows for a variety of detection methods to be employed. Briefly, a primary screening test (i.e., IF and/or selective isolation) is conducted with extracts from vascular tissue sampled from 200 tubers per 25-tonne lot. To confirm th...

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“…A third level of discrimination against false positive results (after the cDNA synthesis and the PCR reaction) is thus ensured by the ELOSA step. Other gel-free methods have been proposed for the detection of PCR amplification products, and notably those based on the fluorogenic 5% nuclease assay (Schoen et al, 1996). In these methods, the amplification products are detected in real time during the PCR, thus speeding up the detection procedure.…”

Section: Discussionmentioning

confidence: 99%

“…More sensitive techniques thus represent a possible alternative. In this respect, RT-PCR tests combining a cDNA synthesis and a PCR amplification have been described for the detection of potato viruses like PVY O , PVY NTN (Weidemann and Maiss, 1996), PLRV (Schoen et al, 1996;Singh et al, 1995) or PVA (Cerovska et al, 1998;.…”

Section: Introductionmentioning

confidence: 99%

RT-PCR-ELOSA tests on pooled sample units for the detection of virus Y in potato tubers

Chandelier

Dubois

Baelen

et al. 2001

Journal of Virological Methods

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A highly sensitive RT-PCR protocol able to detect potato virus Y (PVY) in pooled sample units (tubers) was developed. PVY-specific primers selected in the coat protein gene were found to amplify a 359 bp fragment from diluted crude extract of infected tubers. For the detection of the amplification products, a colorimetric detection procedure in microtiter plates was established. The amplicons are hybridized between a covalently linked capture probe and a specific biotinylated detection probe ELOSA tests. This detection method detects at least 50 pg of virus per reaction for the four cultivars tested. The RT-PCR-ELOSA assay was adapted to pooled units in order to increase the sample size while reducing the number of tests.

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Detection of Potato Leafroll Virus in Dormant Potato Tubers by Immunocapture and a Fluorogenic 5 prime Nuclease RT-PCR Assay

Cited by 70 publications

References 0 publications

Real-time quantitative PCR: a new technology to detect and study phytopathogenic and antagonistic fungi

Real-time quantitative PCR: a new technology to detect and study phytopathogenic and antagonistic fungi

Detection of Ralstonia solanacearum Strains with a Quantitative, Multiplex, Real-Time, Fluorogenic PCR (TaqMan) Assay

RT-PCR-ELOSA tests on pooled sample units for the detection of virus Y in potato tubers

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