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Applied and Environmental Microbiology, June 2006, p. 4256-4263, Vol. 72, No. 6
0099-2240/06/$08.00+0     doi:10.1128/AEM.02706-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Internally Controlled Real-Time PCR Method for Quantitative Species-Specific Detection and vapA Genotyping of Rhodococcus equi{dagger}

David Rodríguez-Lázaro,1 Deborah A. Lewis,1 Alain A. Ocampo-Sosa,1,3 Ursula Fogarty,3 László Makrai,4 Jesús Navas,5 Mariela Scortti,1,2 Marta Hernández,1 and José A. Vázquez-Boland1,2*

Bacterial Molecular Pathogenesis Group, Faculty of Medical and Veterinary Sciences, University of Bristol, Langford, United Kingdom,1 Facultad de Veterinaria, Universidad de León, León, Spain,2 Irish Equine Centre, Johnstown, Naas, Ireland,3 Department of Microbiology and Infectious Diseases, Faculty of Veterinary Science, Budapest, Hungary,4 Departamento de Biología Molecular, Facultad de Medicina, Universidad de Cantabria, Santander, Spain5

Received 15 November 2005/ Accepted 11 April 2006

We developed a novel quantitative real-time PCR (Q-PCR) method for the soil actinomycete Rhodococcus equi, an important horse pathogen and emerging human pathogen. Species-specific quantification was achieved by targeting the chromosomal monocopy gene choE, universally conserved in R. equi. The choE Q-PCR included an internal amplification control (IAC) for identification of false negatives. A second Q-PCR targeted the virulence plasmid gene vapA, carried by most horse isolates but infrequently found in isolates from other sources. The choE-IAC and vapA assays were 100% sensitive and specific as determined using 178 R. equi isolates, 77 nontarget bacteria, and a panel of 60 R. equi isolates with known vapA+ and vapA-negative (including vapB+) plasmid genotypes. The vapA+ frequency among isolate types was as follows: horse, 85%; human, 20%; bovine and pig, 0%; others, 27%. The choE-IAC Q-PCR could detect up to one genome equivalent using R. equi DNA or 100 bacteria/ml using DNA extracted from artificially contaminated horse bronchoalveolar lavage (BAL) fluid. Quantification was linear over a 6-log dynamic range down to {approx}10 target molecules (or 1,000 CFU/ml BAL fluid) with PCR efficiency E of >0.94. The vapA assay had similar performance but appeared unsuitable for accurate (vapA+) R. equi quantification due to variability in target gene or plasmid copy number (1 to 9). The dual-reaction Q-PCR system here reported offers a useful tool to both medical and veterinary diagnostic laboratories for the quantitative detection of R. equi and (optional) vapA+ "horse-pathogenic" genotype determination.

* Corresponding author. Mailing address: Veterinary Molecular Microbiology Section, Faculty of Medical and Veterinary Sciences, University of Bristol, Langford BS40 5DU, United Kingdom. Phone: 44 (0) 117 928 9615. Fax: 44 (0) 117 928 9505. E-mail: v.boland{at}bristol.ac.uk.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, June 2006, p. 4256-4263, Vol. 72, No. 6
0099-2240/06/$08.00+0     doi:10.1128/AEM.02706-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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