Multiplex TaqMan(®) Quantitative PCR Assays for Host-Tick-Pathogen Studies Using the Guinea Pig-Tick-Rickettsia System

Spotted Fever Rickettsiosis (SFR) is caused by spotted fever group Rickettsia spp. (SFGR), and is associated with symptoms common to other illnesses, making it challenging to diagnose before detecting SFGR-specific antibodies. The guinea pig is a valuable biomedical model for studying Spotted Fever Rickettsiosis (SFR); its immune system is more like the human immune system than that of the murine model, and guinea pigs develop characteristic clinical signs. Thus, we have a compelling interest in developing, expanding, and optimizing tools for use in our guinea pig-Amblyomma-Rickettsia system for understanding host-tick-pathogen interactions. With the design and optimization of the three multiplex TaqMan(®) qPCR assays described here, we can detect the two SFGR, their respective primary Amblyomma sp. vectors, and the guinea pig model as part of controlled experimental studies using tick-transmission of SFGR to guinea pigs. We developed qPCR assays that reliably detect each specific target down to 10 copies by producing plasmid standards for each assay target, optimizing the individual primer-probe sets, and optimizing the final multiplex reactions in a methodical, stepwise fashion. We anticipate that these assays, currently designed for in vivo studies, will serve as a foundation for optimal SFGR detection in other systems, including fieldwork.