Nucleic acid amplification for rapid detection of Rhodococcus equi in equine blood and tracheal wash fluids.

The research article investigates the use of nucleic acid amplification techniques, particularly the polymerase chain reaction (PCR), to detect a specific type of bacterium, Rhodococcus equi, in horses and differentiates between the virulent and avirulent strains of this bacterium.

Methodology

• The evaluated method for detection involves the use of nucleic acid amplification techniques. Blood anticoagulated with EDTA and tracheal wash fluid samples were taken from healthy horses.
• These samples were mixed with logarithmic dilutions of virulent (harmful) and avirulent (non-harmful) strains of R equi.
• The DNA was then extracted from these samples and amplified using PCR, a technique that makes millions of copies of a specific DNA sequence, making it easier to detect.
• The primers used for this PCR were specific for the 16S ribosomal subunit gene and the virulence plasmid of R equi. These primers target only the specified regions of DNA to be copied, hence enabling the differentiation of virulent and avirulent strains of the bacteria.

Results

• After performing PCR with 16S ribosomal subunit primers, they could amplify a specific 441-bp segment of DNA from both virulent and avirulent strains of R equi. They found that there was no DNA amplification from samples containing other species of bacteria, suggesting the specificity of the chosen PCR primers.
• Further, the virulence plasmid primers were noted to amplify an 875-bp segment of DNA only from virulent strains of R equi, and not from avirulent R equi or other bacterial species.
• It was identified that virulent strains of R equi could be detected and differentiated from avirulent strains within 12 to 24 hours after sample collection, even with as few as 10 to 100 organisms present.

Conclusion

• The study concludes that PCR can be used to rapidly and accurately identify R equi in equine blood and tracheal wash fluid samples and, importantly, can differentiate between virulent and avirulent strains of this organism.
• Since PCR can provide a diagnosis of R equi infection more rapidly and specifically than using standard culture techniques, applying this assay to soil and fecal samples could prove beneficial in epidemiologic studies and studies of environmental disinfection or decontamination.