Internally controlled real-time PCR method for quantitative species-specific detection and vapA genotyping of Rhodococcus equi

David Rodríguez-Lázaro, Deborah A Lewis, Alain A Ocampo-Sosa, Ursula Fogarty, Laszlo Makrai, Jesus Navas, Mariela Scortti, Marta Hernandez, Jose A Vazquez-Boland

Research output: Contribution to journalArticlepeer-review

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.
Original languageEnglish
Pages (from-to)4256-63
Number of pages8
JournalApplied and Environmental Microbiology
Volume72
Issue number6
Publication statusPublished - Jul 2006

Keywords

  • Animals
  • Bacterial Proteins/genetics
  • Base Sequence
  • DNA Primers
  • Genotype
  • Horses/microbiology
  • Humans
  • Polymerase Chain Reaction/methods
  • Rhodococcus equi/genetics
  • Rhodococcus equi/isolation & purification
  • Sensitivity and Specificity
  • Soil Microbiology
  • Swine/microbiology
  • Virulence Factors/genetics

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