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Home / Equine Research Bank / Appl Environ Microbiol / Internally controlled real-time PCR method for quantitative ...
Applied and environmental microbiology2006; 72(6); 4256-4263; doi: 10.1128/AEM.02706-05

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.
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Publication Date: 2006-06-06 PubMed ID: 16751540PubMed Central: PMC1489618DOI: 10.1128/AEM.02706-05Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research paper discusses the creation of a new quantitative real-time PCR method for the detection and genotyping of Rhodococcus equi, a soil bacterium that can cause disease in horses and humans. The method can identify and quantify this bacterium, even in trace amounts, and enables the researchers to distinguish between variations of the bacterium that are harmful to horses.

Approach and Method

  • The researchers used a method to develop a specific quantitative real-time PCR (Q-PCR), a type of test for detecting and quantifying DNA.
  • To specifically target Rhodococcus equi, they focused on the gene choE, which is conserved in this species. The Q-PCR included an internal amplification control (IAC) to detect any false negatives.
  • A second Q-PCR was designed to single out the virulence plasmid gene vapA, found mainly in horse isolates but rarely elsewhere.

Results

  • The tests were proven to be 100% sensitive and specific when tested on 178 R. equi isolates, 77 nontarget bacteria, and a panel of 60 R. equi isolates.
  • The vapA+ frequency among different isolate types varied; for horse isolates it was 85%, in human it was 20%, while it was not found in bovine and pig isolates.
  • The choE-IAC Q-PCR could detect small amounts up to one genome equivalent and was able to detect 100 bacteria/ml when tested with DNA extracted from artificially contaminated horse bronchoalveolar lavage fluid.

Limits and Future Application

  • The method can quantify over a 6-log dynamic range, down to approximately 10 target molecules (or 1,000 CFU/ml BAL fluid), with a PCR efficiency of >0.94.
  • However, the vapA assay could vary due to changes in the target gene or plasmid copy number (1 to 9) so it might not be ideal for accurate quantification of vapA+ R. equi.
  • In conclusion, the Q-PCR method can be a beneficial tool for medical and veterinary diagnostic labs to quantitatively detect R. equi and also to determine the presence of the “horse-pathogenic” genotype.

Cite This Article

APA
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. Appl Environ Microbiol, 72(6), 4256-4263. https://doi.org/10.1128/AEM.02706-05

Publication

Applied and environmental microbiology
ISSN: 0099-2240
NlmUniqueID: 7605801
Country: United States
Language: English
Volume: 72
Issue: 6
Pages: 4256-4263

Researcher Affiliations

Rodríguez-Lázaro, David
  • Bacterial Molecular Pathogenesis Group, Faculty of Medical and Veterinary Sciences, University of Bristol, Langford, United Kingdom.
Lewis, Deborah A
    Ocampo-Sosa, Alain A
      Fogarty, Ursula
        Makrai, László
          Navas, Jesús
            Scortti, Mariela
              Hernández, Marta
                Vázquez-Boland, José A

                  MeSH Terms

                  • 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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