NUCLEIC-ACID AMPLIFICATION FOR RAPID DETECTION OF RHODOCOCCUS-EQUI INEQUINE BLOOD AND TRACHEAL WASH FLUIDS

Objective-To evaluate the ability of nucleic acid amplification techni ques to detect Rhodococcus equi in equine buffy coat, blood, and trach eal wash fluid and to differentiate between virulent and avirulent str ains of the bacteria. Sample population-Blood anticoagulated with EDTA and tracheal wash fluid from healthy horses. Procedure-Logarithmic di lutions of virulent and avirulent strains of R equi were added to equi ne buffy coat and tracheal wash fluid samples. The DNA was extracted a nd amplified by polymerase chain reaction (PCR), using primers specifi c for the 16S ribosomal subunit gene and the virulence plasmid of R eq ui. Results-PCR with 16S ribosomal subunit primers amplified a 441-bp segment of DNA from virulent and avirulent strains of R equi, but not from samples containing other species of bacteria. The virulence plasm id primers amplified an 875-bp segment of DNA from virulent strains of R equi, but not from avirulent R equi, or from other species of bacte ria. Virulent strains of R equi could be identified by PCR and differe ntiated from avirulent strains within 12 to 24 hours after sample coll ection, with as few as 10 to 100 organisms present. Conclusions-PCR ca n be used to rapidly and accurately identify R equi in equine blood an d tracheal wash fluid samples and can differentiate between virulent a nd avirulent strains of the organism. Clinical Relevance-Because PCR c an confirm a diagnosis of R equi infection in horses more rapidly and specifically than use of standard culture techniques, extrapolation of this assay to soil and fecal samples could be useful in epidemiologic studies and studies of environmental disinfection or decontamination.