Development of a Diagnostic Test Based on the Polymerase Chain Reaction (PCR) to Identify Strains of R. solanacearum Exhibiting the Biovar 2 Genotype

Fegan, Mark; Holoway, G.; Hayward, Andrew; Jn, Timmis

doi:10.1007/978-3-662-03592-4_5

Cited by 55 publications

(47 citation statements)

References 10 publications

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“…The results presented above demonstrate the development of a fluorogenic 5Ј nuclease PCR-based (TaqMan) assay capable of detection and identification of R. solanacearum directly in potato tuber tissue with sensitivity and specificity equal to or greater than those achievable with existing PCR protocols (8,9,24). In routine laboratory studies, R. solanacearum can be detected in conventional PCR assays in aqueous suspensions ranging from 10 (9).…”

Section: Discussionmentioning

confidence: 85%

“…In routine laboratory studies, R. solanacearum can be detected in conventional PCR assays in aqueous suspensions ranging from 10 (9). However in potato extracts, detection limits for the former assay (24) have been shown to be increased to 10 6 cells per ml or higher (8).…”

Section: Discussionmentioning

confidence: 99%

“…The lower C T values obtained from the RS-P assay, rather than those obtained from B2-P with the same extracts, indicate a higher concentration of RS target DNA than of B2 target DNA. This may be explained by RS target DNA being part of the multicopy 16S rRNA gene compared with the B2 target DNA, whose genomic location and function are not known (9).…”

Section: Discussionmentioning

confidence: 99%

“…The broad-host-range R. solanacearum probe (RS-P) is partially homologous to 16S rRNA gene primer OLI1 (24), with primers (RS-I and RS-II) flanking this region. The primers and TaqMan probe for the biovar 2A-specific assay were elucidated from the biovar 2A-specific DNA sequence described by Fegan et al (9), retaining the original forward primer 631 (B2-II) and selecting the probe (B2-P) and reverse primer (B2-I) from the upstream region. The internal positive control primer-probe combination was designed according to the published sequence (20) of the abundant constitutive cytochrome oxidase (COX) gene.…”

Section: Methodsmentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 85%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

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

View full text Add to dashboard Cite

show abstract

“…Several PCR protocols and specific primers were designed for detection or identification of the species and for phylotyping ( Table 2) (SEAL et al, 1993;ELPHINSTONE et al, 1996;OPINA et al, 1997;FEGAN et al, 1998;BOUDAZIN et al, 1999;PASTRIK;MAISS, 2000;LUISETTI, 2000;WELLER et al, 2000). Classification into phylotypes is done by PCR Multiplex with the Nmult series primers (based on ITS region) and the classification into sequevar by partially sequencing gene egl (encoding the enzyme endoglucanase).…”

Section: Genetic Diversity Of Ralstonia Solanacearum In Brazilmentioning

confidence: 99%

Occurrence and diversity of Ralstonia solanacearum populations in Brazil

et al. 2015

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ABSTRACT:Ralstonia solanacearum is a gram-negative soil-borne bacterium capable of infection of hundreds of vegetable species over more than 50 botanical families, causing bacterial wilt, except for bananas, when the disease is called Moko. It deserves special attention, from all other plant pathogenic bacteria, for its high phenotypic and genotypic plasticity, a characteristic that makes disease control extremely difficult. In this context, frequent and necessary surveys are conduct in the attempt of characterizing the prevailing strains of R. solanacearum in each region where the disease has been reported. However, knowledge about occurrence and diversity of R. solanacearum in Brazil is fragmented and in some cases, based on inconclusive studies with few strains, little representative of a given region. The need to obtain a greater picture guided this review. The occurrence of this bacterium in Brazilian States and the possible causes for its dissemination are presented, with emphasis on studies of genetic variability of populations of R. solanacearum in the country. The compiled results report a wide distribution of the bacterium in Brazil and great variability of its populations among locations. Partly due to the difficulty of detecting small titer of bacteria in samples, paucity of information about the origin of inoculum in certain regions is observed, as well as the need for detecting the presence of the pathogen in asymptomatic plants, potato tubers with latent infections, soil, and water, which are the major causes of bacterial dissemination into areas without any disease history.

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