Figure 5

Inclusion of three tick-borne pathogens in the presence of human DNA in a single quadruplex assay does not affect the sensitivity of their detection. Conditions for a quadruplex PCR assay were optimized such that eight primers and four different molecular beacons for respective amplicons were present in the same tube along with the other reagents required for the PCR. Sensitivity of detection of two bacterial pathogens, extracellular spirochete B. burgdorferi (A) and obligate intracellular pathogen A. phagocytophilum (C) , along with the intracellular parasite, B. microti (B), was not affected in this quadruplex assay, indicating that the assay can be extended for simultaneous diagnosis of all three tick-borne pathogens in the patients, especially in the endemic regions. Detection of the ACTA1 amplicon in the same reaction will offer as control for human DNA (D) and quality of DNA preparation when the patient samples will be used for diagnosis of the infecting organism.