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.


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.
Harrington
Jessica R
JR
Department of Large Animal Medicine, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843, USA.
Golding
Michael C
MC
Martens
Ronald J
RJ
Halbert
Natalie D
ND
Cohen
Noah D
ND
D000193:Actinomycetales Infections / Q000175:diagnosis / Q000662:veterinary*; D000818:Animals; D001992:Bronchoalveolar Lavage Fluid / Q000382:microbiology; D004269:DNA, Bacterial / Q000032:analysis; D006734:Horse Diseases / Q000175:diagnosis / Q000382:microbiology*; D006736:Horses; D016133:Polymerase Chain Reaction / Q000379:methods*; D016929:Rhodococcus equi / Q000302:isolation & purification* / Q000472:pathogenicity*; D012680:Sensitivity and Specificity; D014774:Virulence
D004269:DNA, Bacterial