Clostridium difficile: A difficult diagnosis made possible by real-time PCR

 Diagnosing the underlying cause of diarrhea and enterocolitis in horses is difficult. One bacterium that is associated with enterocolitis in foals is the anaerobic bacterium, Clostridium difficile. This bacterium is difficult to culture, thus other diagnostic methods must be used. A real-time PCR assay for the Toxin A and Toxin B genes of Clostridium difficile was developed and validated for more efficient diagnosis of this bacterium from clinical samples. Four sets of primers (two for Toxin A and two for Toxin B) were evaluated. Toxin A Set 5 and Toxin B Set 2 were determined to be most effective at detection the respective toxin genes. We used Sanger sequencing to confirm that the amplicon was the correct gene target, and then optimized the PCR protocol for maximum sensitivity and efficiency. PCR parameters included annealing time and temperature, primer concentration, and probe concentration. We used different fluorophores on the probes, to be able to multiplex this PCR reaction; FAM for Toxin A, HEX for Toxin B and Cy5 for the internal control. However when a multiplex reaction was attempted, there was interference. Thus the final assay includes a separate reactions for each toxin gene. A serial dilution of a positive C. diff ATCC control (BAA-1870DQ Quantitative Genomic DNA from Clostridium difficile Strain 4118) was used to test the efficiencies which were determined to be 91.3% for Toxin A and 97.6% for Toxin B. Validation was performed using thirty negative sample controls, while positive sample validation will be ongoing.