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, L. Makrai, Jesús Navas, Mariela Scortti, Marta Hernández, José A. Vázquez-Boland

Research output: Contribution to journalArticle

34 Citations (Scopus)

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 monocepy 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.

Original languageEnglish
Pages (from-to)4256-4263
Number of pages8
JournalApplied and Environmental Microbiology
Volume72
Issue number6
DOIs
Publication statusPublished - Jun 2006

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Rhodococcus equi
horse
genotyping
quantitative analysis
Real-Time Polymerase Chain Reaction
quantitative polymerase chain reaction
Horses
plasmid
amplification
horses
plasmids
gene
Plasmids
genotype
Bronchoalveolar Lavage Fluid
pathogen
assay
DNA
bacterium
fluid

ASJC Scopus subject areas

  • Environmental Science(all)
  • Biotechnology
  • Microbiology

Cite this

Internally controlled real-time PCR method for quantitative species-specific detection and vapA genotyping of Rhodococcus equi. / Rodríguez-Lázaro, David; Lewis, Deborah A.; Ocampo-Sosa, Alain A.; Fogarty, Ursula; Makrai, L.; Navas, Jesús; Scortti, Mariela; Hernández, Marta; Vázquez-Boland, José A.

In: Applied and Environmental Microbiology, Vol. 72, No. 6, 06.2006, p. 4256-4263.

Research output: Contribution to journalArticle

Rodríguez-Lázaro, D, Lewis, DA, Ocampo-Sosa, AA, Fogarty, U, Makrai, L, Navas, J, Scortti, M, Hernández, M & Vázquez-Boland, JA 2006, 'Internally controlled real-time PCR method for quantitative species-specific detection and vapA genotyping of Rhodococcus equi', Applied and Environmental Microbiology, vol. 72, no. 6, pp. 4256-4263. https://doi.org/10.1128/AEM.02706-05
Rodríguez-Lázaro, David ; Lewis, Deborah A. ; Ocampo-Sosa, Alain A. ; Fogarty, Ursula ; Makrai, L. ; Navas, Jesús ; Scortti, Mariela ; Hernández, Marta ; Vázquez-Boland, José A. / Internally controlled real-time PCR method for quantitative species-specific detection and vapA genotyping of Rhodococcus equi. In: Applied and Environmental Microbiology. 2006 ; Vol. 72, No. 6. pp. 4256-4263.
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