A TaqMan-PCR protocol for quantification and differentiation of the phytopathogenic Clavibacter michiganensis subspecies

Bach, H.-J.; Jessen, I; Schloter, Michael; Munch, Jean Charles

doi:10.1016/s0167-7012(02)00152-5

Cited by 56 publications

(49 citation statements)

References 12 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The theoretical quantification limit was determined through the calculation of the expected number of template molecules at each dilution with the P value as 0.05 (the calculations were performed assuming a binomial distribution and confirmed by Monte Carlo simulations) and establishing as the theoretical quantification limit the lowest sample dilution in which the 95% confidence interval does not overlap with that of the next dilution (Table 1). This value is identical to that previously reported for the corresponding uniplex assay (18) and similar to that reported for other quantitative Q-PCR systems (4,12,13,16,21).…”

supporting

confidence: 91%

A Novel Real-Time PCR for Listeria monocytogenes That Monitors Analytical Performance via an Internal Amplification Control

Rodrı́guez-Lázaro

Pla

Scortti

et al. 2005

Appl Environ Microbiol

View full text Add to dashboard Cite

We describe a novel quantitative real-time (Q)-PCR assay for Listeria monocytogenes based on the coamplification of a target hly gene fragment and an internal amplification control (IAC). The IAC is a chimeric double-stranded DNA containing a fragment of the rapeseed BnACCg8 gene flanked by the hly-specific target sequences. This IAC is detected using a second TaqMan probe labeled with a different fluorophore, enabling the simultaneous monitoring of the hly and IAC signals. The hly-IAC assay had a specificity and sensitivity of 100%, as assessed using 49 L. monocytogenes isolates of different serotypes and 96 strains of nontarget bacteria, including 51 Listeria isolates. The detection and quantification limits were 8 and 30 genome equivalents, and the coefficients for PCR linearity (R 2 ) and efficiency (E) were 0.997 and 0.80, respectively. We tested the performance of the hly-IAC Q-PCR assay using various broth media and food matrices. Fraser and half-Fraser media, raw pork, and raw or cold-smoked salmon were strongly PCR-inhibitory. This Q-PCR assay for L. monocytogenes, the first incorporating an IAC to be described for quantitative detection of a food-borne pathogen, is a simple and robust tool facilitating the identification of false negatives or underestimations of contamination loads due to PCR failure.

show abstract

supporting

confidence: 91%

A Novel Real-Time PCR for Listeria monocytogenes That Monitors Analytical Performance via an Internal Amplification Control

Rodrı́guez-Lázaro

Pla

Scortti

et al. 2005

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…Real-time PCR reduces staff input and time, and lowers the risk of false-positive results due to carryover contamination. In previous studies, real-time PCR protocols using the TaqMan fluorescent chemistry methodology were developed for the single detection of R. solanacearum (Ozakman and Schaad 2003;Weller et al 2000) or of C. michiganensis sepedonicus (Bach et al 2003;Schaad et al 1999).…”

Section: Introductionmentioning

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

“…For this purpose, an external standard curve was drawn by serial dilution of strain O cells in KPB buffer from washed untreated apples. In comparison with serial dilution of genomic DNA (Filion et al, 2003a), plasmid standard (Bach et al, 2003) or cells (Bowers et al, 2000) in sterile water, this protocol fits better to the whole sample processing and takes into account the unavoidable minimal inaccuracies occurring during DNA extraction and manipulation (e.g. presence of PCR inhibitors).…”

Section: Discussionmentioning

confidence: 99%

Development of real-time PCR using Minor Groove Binding probe to monitor the biological control agent Candida oleophila (strain O)

Massart

Clercq

Salmon

et al. 2005

Journal of Microbiological Methods

View full text Add to dashboard Cite

A real-time PCR assay using a 3V -Minor Groove Binding (MGB) probe was developed for specific detection and monitoring of Candida oleophila (strain O), a biocontrol agent against Botrytis cinerea and Penicillium expansum, on harvested apples. The application of the RAPD technique on C. oleophila strains followed by reproducible sequence characterized amplified region (SCAR) amplifications allowed the identification of a semi-specific fragment of 244 bp, observed in the profiles of strain O and three other C. oleophila strains. After sequencing, polymorphisms (3%) were observed between the strain O sequence and the three other sequences. A 3V -Minor Groove Binding probe was designed to specifically match a region of the strain O sequence and was able to discriminate a single base mutation or a two-base difference in the corresponding sequences of the non-target strains. This specific detection method was applied to monitor strain O population, recovered by a washing buffer, from harvested apples. Population densities were calculated using an external standard curve consisting in a serial dilution of strain O cells in the washing buffer from untreated apples. Linearity in the standard curve was kept between 1.64Â10 2 and 1.64Â10 5 cfu cm À2 of apple surface. During a first practical experiment, the calculated population densities were similar to those obtained by plating on semi-selective media. This new real-time PCR method is a promising tool to monitor quickly and specifically strain O population on apple surface in middle-or large-scale experiments. D

show abstract

A TaqMan-PCR protocol for quantification and differentiation of the phytopathogenic Clavibacter michiganensis subspecies

Cited by 56 publications

References 12 publications

A Novel Real-Time PCR for Listeria monocytogenes That Monitors Analytical Performance via an Internal Amplification Control

A Novel Real-Time PCR for Listeria monocytogenes That Monitors Analytical Performance via an Internal Amplification Control

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 real-time PCR using Minor Groove Binding probe to monitor the biological control agent Candida oleophila (strain O)

Contact Info

Product

Resources

About