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American journal of veterinary research2005; 66(5); 755-761; doi: 10.2460/ajvr.2005.66.755

Evaluation of a real-time quantitative polymerase chain reaction assay for detection and quantitation of virulent Rhodococcus equi.

Abstract: To evaluate a real-time quantitative polymerase chain reaction (QPCR) assay in the detection and quantitation of virulent Rhodococcus equi. Methods: 1 virulent, 2 intermediately virulent, and 2 avirulent strains of R. equi and 16 isolates of bacteria genetically related to R. equi. Methods: The QPCR assay was evaluated for detection and quantitation of the virulence-associated gene (vapA) of R. equi in pure culture and in samples of tracheobronchial fluid, which were inoculated with known numbers of virulent R. equi. Results were compared with those derived via quantitative microbial culture and standard polymerase chain reaction methods. Results: The QPCR assay detected the vapA gene in pure culture of R. equi and in tracheobronchial fluid samples that contained as few as 20 CFUs of virulent R. equi/mL and accurately quantitated virulent R. equi to 10(3) CFUs/mL of fluid. The assay was highly specific for detection of the vapA gene of virulent R. equi and was more sensitive than standard polymerase chain reaction for detection of R. equi in tracheobronchial fluid. Conclusions: The QPCR assay appears to be a rapid and reliable method for detecting and quantitating virulent R. equi. The accuracy of the QPCR assay is comparable to that of quantitative microbial culture. The increased sensitivity of the QPCR method in detection of virulent R. equi should facilitate rapid and accurate diagnosis of R. equi pneumonia in foals.
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Publication Date: 2005-06-09 PubMed ID: 15940818DOI: 10.2460/ajvr.2005.66.755Google Scholar: Lookup
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  • Evaluation Study
  • Journal Article
  • Research Support
  • Non-U.S. Gov't

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.

This research tested a real-time quantitative polymerase chain reaction (QPCR) assay for detecting and measuring amounts of a bacteria called virulent Rhodococcus equi. The study found that the QPCR assay is a quick and reliable method that can detect even small amounts of the bacteria and could aid in accurately diagnosing diseases like foal pneumonia.

Objective and Methodology

  • The main goal of this research was to assess the effectiveness of a real-time quantitative polymerase chain reaction (QPCR) assay in detecting and quantifying virulent Rhodococcus equi, a bacteria that can cause diseases like pneumonia in young horses.
  • The study used one virulent strain, two intermediately virulent strains, and two avirulent strains of R. equi, along with 16 isolates of bacteria genetically related to R. equi.
  • The QPCR assay’s task was to identify and measure the amount of virulence-associated gene (vapA) of R. equi in pure culture and in tracheobronchial fluid samples inoculated with this bacteria.
  • Its results were then compared with those derived from quantitative microbial culture and standard polymerase chain reaction methods.

Results and Findings

  • The QPCR assay could detect the vapA gene in pure culture of R. equi and in tracheobronchial fluid samples containing as few as 20 Colony Forming Units (CFUs) per mL of virulent R. equi.
  • In addition, the method was able to accurately quantify the number of virulent R. equi to 103 CFUs/mL of fluid.
  • It was highly specific for detecting the vapA gene and showed greater sensitivity than standard polymerase chain reaction in detecting R. equi in tracheobronchial fluid.

Conclusion and Applications

  • The QPCR assay proved to be quick and efficient in detecting and quantifying virulent R. equi.
  • Its accuracy is on par with that of the traditional quantitative microbial culture method.
  • The assay’s enhanced sensitivity over standard detection methods can potentially lead to quicker and more precise diagnosis of diseases caused by R. equi, like foal pneumonia.

Cite This Article

APA
Harrington JR, Golding MC, Martens RJ, Halbert ND, Cohen ND. (2005). Evaluation of a real-time quantitative polymerase chain reaction assay for detection and quantitation of virulent Rhodococcus equi. Am J Vet Res, 66(5), 755-761. https://doi.org/10.2460/ajvr.2005.66.755

Publication

American journal of veterinary research
ISSN: 0002-9645
NlmUniqueID: 0375011
Country: United States
Language: English
Volume: 66
Issue: 5
Pages: 755-761

Researcher Affiliations

Harrington, Jessica R
  • Department of Large Animal Medicine, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843, USA.
Golding, Michael C
    Martens, Ronald J
      Halbert, Natalie D
        Cohen, Noah D

          MeSH Terms

          • Actinomycetales Infections / diagnosis
          • Actinomycetales Infections / veterinary
          • Animals
          • Bronchoalveolar Lavage Fluid / microbiology
          • DNA, Bacterial / analysis
          • Horse Diseases / diagnosis
          • Horse Diseases / microbiology
          • Horses
          • Polymerase Chain Reaction / methods
          • Rhodococcus equi / isolation & purification
          • Rhodococcus equi / pathogenicity
          • Sensitivity and Specificity
          • Virulence

          Citations

          This article has been cited 6 times.
          1. Sting R, Schwabe I, Kieferle M, Münch M, Rau J. Fatal Infection in an Alpaca (Vicugna pacos) Caused by Pathogenic Rhodococcus equi. Animals (Basel) 2022 May 19;12(10).
            doi: 10.3390/ani12101303pubmed: 35625149google scholar: lookup
          2. Narváez SÁ, Fernández I, Patel NV, Sánchez S. Novel Quantitative PCR for Rhodococcus equi and Macrolide Resistance Detection in Equine Respiratory Samples. Animals (Basel) 2022 May 3;12(9).
            doi: 10.3390/ani12091172pubmed: 35565598google scholar: lookup
          3. Madrigal RG, Shaw SD, Witkowski LA, Sisson BE, Blodgett GP, Chaffin MK, Cohen ND. Use of Serial Quantitative PCR of the vapA Gene of Rhodococcus equi in Feces for Early Detection of R. equi Pneumonia in Foals. J Vet Intern Med 2016 Mar-Apr;30(2):664-70.
            doi: 10.1111/jvim.13828pubmed: 26806422google scholar: lookup
          4. Stefańska I, Witkowski L, Rzewuska M, Dzieciątkowski T. Development and evaluation of the internal-controlled real-time PCR assay for Rhodococcus equi detection in various clinical specimens. J Vet Med Sci 2016 May 3;78(4):543-9.
            doi: 10.1292/jvms.15-0516pubmed: 26655770google scholar: lookup
          5. Shaw SD, Cohen ND, Chaffin MK, Blodgett GP, Syndergaard M, Hurych D. Estimating the Sensitivity and Specificity of Real-Time Quantitative PCR of Fecal Samples for Diagnosis of Rhodococcus equi Pneumonia in Foals. J Vet Intern Med 2015 Nov-Dec;29(6):1712-7.
            doi: 10.1111/jvim.13631pubmed: 26436545google scholar: lookup
          6. Rodríguez-Lázaro D, Lewis DA, Ocampo-Sosa AA, Fogarty U, Makrai L, Navas J, Scortti M, Hernández M, Vázquez-Boland JA. Internally controlled real-time PCR method for quantitative species-specific detection and vapA genotyping of Rhodococcus equi. Appl Environ Microbiol 2006 Jun;72(6):4256-63.
            doi: 10.1128/AEM.02706-05pubmed: 16751540google scholar: lookup
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