Furthermore, with the recent emergence of ticks infected with deer tick virus and Powassan virus lineages in New York and Connecticut in the United States and several European countries [87–89], it will be useful to include an assay for their diagnosis. Our assay could easily be extended
to include the most prevalent virus amplicon after an addition reverse transcription step. Since most real-time PCR machines are capable of detecting five fluorophore with non-overlapping spectrofluorometric spectra and we have only used four in our assay, we anticipate that achieving this goal will be relatively simple. In summary, the ability of the assay GANT61 supplier described here to detect multiple tick-borne pathogens simultaneously will be a boon for health professionals to design more effective treatment regimes for coinfections
when this assay is approved for mass application. Conclusions Optimized conditions and PCR parameters, including the amplicons of the conserved genes present in Lyme spirochetes, A. phagocytophilum and the tick-borne parasite B. microti, and molecular beacon probes tagged with distinct fluorophores, can detect all three pathogens in a sensitive manner. Excessive presence of any pathogen did not affect sensitivity of detection of the other pathogen present in lower dose. The real-time PCR assay described here can be used both; to detect coinfections with more than one tick-borne pathogen in the endemic regions of the USA and the European countries as well as to detect each pathogen individually with equal efficiency. Since transfusion-associated babesiosis cases and fatalities are increasing steadily, Cisplatin research buy the assay can also be used for detection of Babesia species and A. phagocytophilum in blood donated to the blood banks after minor modifications. The assay will be used in the future for diagnosis of tick-borne
diseases after further optimization with patient samples. Acknowledgements This work was supported by National Institutes of Health Diflunisal grant VX-770 chemical structure R01-AI089921 to NP. SAEM was partly supported by the NIH grant R01-MH-079197. We are grateful to Edouard Vannier of Tufts Medical Center for generously providing B. microti infected mice blood and acknowledge the help from John Leong’s laboratory at Tufts Medical Center in isolating and shipping the genomic DNA to us. We also thank Errol Fikrig of Yale University School of Medicine for generously providing us A. phagocytophilum genomic DNA for this study. References 1. Dantas-Torres F, Chomel BB, Otranto D: Ticks and tick-borne diseases: a One Health perspective. Trends Parasitol 2012,28(10):437–446.PubMedCrossRef 2. Heyman P, Cochez C, Hofhuis A, van der Giessen J, Sprong H, Porter SR, Losson B, Saegerman C, Donoso-Mantke O, Niedrig M, et al.: A clear and present danger: tick-borne diseases in Europe. Expert Rev Anti Infect Ther 2010,8(1):33–50.PubMedCrossRef 3.