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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

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 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.

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