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

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 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 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.