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Updated: Jul 10, 2026
Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control
Published on: March 30, 2015
Evaluation of a real-time quantitative polymerase chain reaction assay for detection and quantitation of virulent
Jessica R Harrington1, Michael C Golding, Ronald J Martens
1Department of Large Animal Medicine, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843, USA.
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.
