Drs. Marras, Tyagi, and Kramer used these probes to distinguish alleles that differ in as little as a single nucleotide

polymorphism (SNPs) [55, 56]. The basis of this extraordinary specificity is that hairpin-shaped probes can assume two different stable states, by: (i) forming double-stranded hybrids with their LY294002 manufacturer target sequence, or (ii) retaining their partially double-stranded structure when not bound to a target. Any mismatch between the probe sequence of the molecular beacon and the target sequence destabilizes the probe-target hybrid, leading to return of the molecular beacon in its stable hairpin structure [57, 58]. Unlike hairpin-shaped probes, linear probes such a TaqMan probes have only one conformation, either on or off the target. This decreases

difference between the melting temperature of a perfectly matched target sequence and a single-nucleotide mismatched target sequence makes discrimination between two scenarios more difficult to discern [58–60]. Furthermore, Taqman probes are digested by the endonuclease activity of the Taq polymerase in each PCR cycle, such that optimization of both annealing and digestion of the probe becomes buy Daporinad more challenging in the development of multiplex assays. Our success in utilizing the extraordinary specificity of molecular beacon probes to detect the recA gene of B. burgdorferi, and to quantitate the number of spirochetes present in infected mouse tissue [61] offered us an incentive to develop the assay for diagnosis of Lyme disease in humans. We have now optimized the assay to work in the presence of human DNA for it to become useful as diagnostic test for human Lyme disease. We describe here expansion of a simplified, highly sensitive multiplex

real-time PCR assay by incorporating specific molecular beacons that can distinguish B. burgdorferi, A. phagocytophilum and B. microti simultaneously. Application of this assay will make a significant difference in achieving the rapid and accurate diagnosis of Lyme disease, anaplasmosis and babesiosis in a cost-effective Ketotifen manner. Methods Microbial strains and human cell line For standardization of conditions for real-time PCR diagnostic assay for Lyme disease, N40 strain clone D10/E9 of B. burgdorferi (sensu stricto), VS461 strain of B. afzelii and PBi strain of B. garinii were grown in BSKII medium supplemented with 6% rabbit serum at 33°C. Dr. Edouard Vannier of Tufts Medical Center at Boston, and Dr. Errol Fikrig of Yale University School of Medicine generously provided the genomic DNA from B. microti strain RM/NS and A. phagocytophilum strain HZ, respectively. Human embryonic kidney 293 cells were cultured in a 1:1 mix of DMEM (low glucose) and Ham’s F12 medium (Life Technologies, NY) supplemented with 10% FBS to isolate human DNA used in the assays. Isolation of B.