A real-time multiplex PCR assay for detection of the causative agents of rat bite fever, Streptobacillus moniliformis and zoonoticStreptobacillus species.

Rat bite fever (RBF) caused by Streptobacillus moniliformis has been described as a diagnostic challenge. While it has a favorable prognosis with treatment, timely diagnosis is hindered by the lack of culture-free identification methods. Here we present a multiplex real-time PCR assay that detects the zoonotic Streptobacillus spp. as well as differentiate the primary causative agent of RBF, Streptobacillus moniliformis. The performance of this assay was evaluated using mock clinical specimens for blood, serum, and urine. Analytical sensitivity was determined to be 3-4 genome equivalents (GE)/µl for the zoonotic Streptobacillus spp. target, and 1-2 GE/µl for the S. moniliformis specific target. The assay correctly detected only the intended targets with no cross-reactivity identified. The pathogen was detected in all spiked matrices and not detected in the negative non-spiked specimens. This rapid diagnostic assay may permit quicker diagnosis of RBF patients.