Detection of Clavibacter michiganensis subsp. sepedonicus by AmpliDet RNA, a new technology based on real time monitoring of NASBA amplicons with a molecular beacon

Beckhoven, J.R.C.M. van; Stead, D. E.; Wolf, J.M. van der

doi:10.1046/j.1365-2672.2002.01765.x

Cited by 37 publications

(23 citation statements)

References 35 publications

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“…In addition, they may amplify the DNA of closely related species and even the DNA of other nonpathogenic soil bacteria (Van der Wolf et al 2005). Other techniques developed for C. michiganensis identification include fluorescent in situ hybridization (FISH) (Van Beuningen et al 1995), real-time PCR (Schaad et al 1999;Bach et al 2003), and nucleic acid sequence based amplification (NASBA) (Van Beckhoven et al 2002). The methods that are applied to differentiate C. michiganensis species are restriction fragment length polymorphisms (RFLP) analysis of the amplified internal transcribed spacer (ITS) between 16S and 23S rRNA genes region of the rrn operon (Lee et al 1997a;b;Borowicz 2001) and rep-PCR (Louws et al 1998).…”

Section: Introductionmentioning

confidence: 99%

Polymorphism analysis of housekeeping genes for identification and differentiation of Clavibacter michiganensis subspecies

Waleron

Kamasa

et al. 2011

Eur J Plant Pathol

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The utility of polymorphism analysis was determined for differentiation of the following subspecies of the Gram-positive plant pathogenic bacterium, Clavibacter michiganensis: C. m. subsp. michiganensis, C. m. subsp. sepedonicus, C. m. subsp. insidiosus C. m. subsp. nebraskensis, and C. m. subsp. tessellarius. Specific primers designed for amplification of the housekeeping genes recA, rpoB, and rpoD generated 827-, 1037-, and 862-bp DNA fragments, respectively. PCR products obtained from 40 C. michiganensis strains were analysed using RFLP with four restriction endonucleases, and those PCR products with specific RFLP patterns were sequenced. The genotypes discriminated after PCR-RFLP were specific for each subspecies and also allowed for differentiation of C. m. subsp. michiganensis strains. Sequence analysis of the recA, rpoB, and rpoD gene fragments also distinguished C. michiganensis subspecies and was useful for phylogenetic analysis of all subspecies. For rapid, inexpensive, and effective differentiation of the five subspecies in this research, we recommend the amplification of recA and/or rpoD gene fragments and digestion of the PCR products with the restriction endonuclease FnuDII.

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Section: Introductionmentioning

confidence: 99%

Polymorphism analysis of housekeeping genes for identification and differentiation of Clavibacter michiganensis subspecies

Waleron

Kamasa

et al. 2011

Eur J Plant Pathol

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“…The isolates must be identified by serology, bioassay and a set of conventional nutritional and physiological tests (Jansen and Van Vaerenbergh, 1987). Since the whole procedure is time consuming, different detection and identification methods based on molecular techniques have been proposed to speed up the process (De Boer et al, 1995;Li et al, 1997;Louws et al, 1998;Wullings et al, 1998;Pastrik and Rainey, 1999;Pastrik, 2000;Palomo et al, 2000;Smith et al, 2001;Beckhoven et al, 2002;Rivas et al, 2002;Fessehaie et al, 2003). Moreover, the European Union has supported a project (DIAGPRO) for the standardization and validation of a protocol to detect and diagnose this quarantine bacterium (available in www.csl.gov.uk/science/organ/ph/diagpro).…”

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confidence: 99%

Evaluation of the API 50CH and API ZYM systems for rapid characterization of Clavibacter michiganensis subsp. sepedonicus, causal agent of potato ring rot

et al. 2006

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API 50CH and API ZYM systems were used to characterize fifty-three strains of Clavibacter michiganensis subsp. sepedonicus from different geographic locations and several reference strains of the same and different species, including other potato pathogens. Clavibacter michiganensis subsp. sepedonicus strains displayed a high level of homogeneity, both in carbohydrate utilization and in enzymatic activity. Using API 50CH and API ZYM it was possible to differentiate C. michiganensis subsp. sepedonicus strains from the remaining taxa analysed in this study, which included representative strains of the other subspecies of C. michiganensis as well as other bacterial pathogens affecting potatoes. Therefore, these systems could be used as an effective method to characterize C. michiganensis subsp. sepedonicus. Such a procedure would constitute an alternative system to the conventional nutritional and physiological identification tests currently included in the official methods employed in the European Union to detect and identify this bacterium. The results obtained with the API systems agreed with the current taxonomic classification of C. michiganensis, clearly separating sepedonicus from the other subspecies belonging to this species.

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“…The oligonucleotides used in this study are shown in Table 1. Primers Af2 and Ar and molecular beacon MB CMS1 (specific sequence) were previously designed and tested by van Beckhoven et al (24) and modified as indicated in Table 1. M. avium subsp.…”

Section: Methodsmentioning

confidence: 99%

Construction Strategy for an Internal Amplification Control for Real-Time Diagnostic Assays Using Nucleic Acid Sequence-Based Amplification: Development and Clinical Application

Rodrı́guez-Lázaro

D’Agostino²,

Pla

et al. 2004

J Clin Microbiol

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An important analytical control in molecular amplification-based methods is an internal amplification control (IAC), which should be included in each reaction mixture. An IAC is a nontarget nucleic acid sequence which is coamplified simultaneously with the target sequence. With negative results for the target nucleic acid, the absence of an IAC signal indicates that amplification has failed. A general strategy for the construction of an IAC for inclusion in molecular beacon-based real-time nucleic acid sequence-based amplification (NASBA) assays is presented. Construction proceeds in two phases. In the first phase, a double-stranded DNA molecule that contains nontarget sequences flanked by target sequences complementary to the NASBA primers is produced. At the 5 end of this DNA molecule is a T7 RNA polymerase binding sequence. In the second phase of construction, RNA transcripts are produced from the DNA by T7 RNA polymerase. This RNA is the IAC; it is amplified by the target NASBA primers and is detected by a molecular beacon probe complementary to the internal nontarget sequences. As a practical example, an IAC for use in an assay for the detection of Mycobacterium avium subsp. paratuberculosis is described, its incorporation and optimization within the assay are detailed, and its application to spiked and natural clinical samples is shown to illustrate the correct interpretation of the diagnostic results.

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Detection of Clavibacter michiganensis subsp. sepedonicus by AmpliDet RNA, a new technology based on real time monitoring of NASBA amplicons with a molecular beacon

Cited by 37 publications

References 35 publications

Polymorphism analysis of housekeeping genes for identification and differentiation of Clavibacter michiganensis subspecies

Polymorphism analysis of housekeeping genes for identification and differentiation of Clavibacter michiganensis subspecies

Evaluation of the API 50CH and API ZYM systems for rapid characterization of Clavibacter michiganensis subsp. sepedonicus, causal agent of potato ring rot

Construction Strategy for an Internal Amplification Control for Real-Time Diagnostic Assays Using Nucleic Acid Sequence-Based Amplification: Development and Clinical Application

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