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

Jessica R. Harrington Department of Large Animal Medicine and Surgery, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843.

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Michael C. Golding Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843.
Present address is Cold Spring Harbor Laboratories, McClintock Building, 1 Bungtown Rd, Cold Spring Harbor, NY 11724.

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Ronald J. Martens Department of Large Animal Medicine and Surgery, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843.

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Natalie D. Halbert Department of Large Animal Medicine and Surgery, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843.

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Noah D. Cohen Department of Large Animal Medicine and Surgery, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843.

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Abstract

Objective—To evaluate a real-time quantitative polymerase chain reaction (QPCR) assay in the detection and quantitation of virulent Rhodococcus equi.

Sample Population—1 virulent, 2 intermediately virulent, and 2 avirulent strains of R equi and 16 isolates of bacteria genetically related to R equi.

Procedure—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 vapAgene 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 103 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 and Clinical Relevance—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. (Am J Vet Res 2005;66:755–761)

Abstract

Objective—To evaluate a real-time quantitative polymerase chain reaction (QPCR) assay in the detection and quantitation of virulent Rhodococcus equi.

Sample Population—1 virulent, 2 intermediately virulent, and 2 avirulent strains of R equi and 16 isolates of bacteria genetically related to R equi.

Procedure—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 vapAgene 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 103 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 and Clinical Relevance—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. (Am J Vet Res 2005;66:755–761)

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