title: Internally controlled real-time PCR method for quantitative species-specific detection and vapA genotyping of Rhodococcus equi.
abstract: 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 approximately 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.
authors.affiliation: Bacterial Molecular Pathogenesis Group, Faculty of Medical and Veterinary Sciences, University of Bristol, Langford, United Kingdom.
authors.forename: David
authors.initials: D
authors.lastname: Rodríguez-Lázaro
authors.forename: Deborah A
authors.initials: DA
authors.lastname: Lewis
authors.forename: Alain A
authors.initials: AA
authors.lastname: Ocampo-Sosa
authors.forename: Ursula
authors.initials: U
authors.lastname: Fogarty
authors.forename: László
authors.initials: L
authors.lastname: Makrai
authors.forename: Jesús
authors.initials: J
authors.lastname: Navas
authors.forename: Mariela
authors.initials: M
authors.lastname: Scortti
authors.forename: Marta
authors.initials: M
authors.lastname: Hernández
authors.forename: José A
authors.initials: JA
authors.lastname: Vázquez-Boland
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers; D005838:Genotype; D006736:Horses / Q000382:microbiology; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D016929:Rhodococcus equi / Q000235:genetics* / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D012988:Soil Microbiology; D013552:Swine / Q000382:microbiology; D037521:Virulence Factors / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D037521:Virulence Factors