Abi 7500 fast real time pcr thermal cycler

SYBR green-based qPCRs were used to detect genomic DNA of pathogens (see Table 1 for primers used and references). Fast SYBR Green Master Mix (cat# 4444432, Applied Biosystems, Foster City, CA) was used in 25 µl reaction following manufacturer’s recommendations. DNA was amplified using an ABI 7500 Fast Real-Time PCR thermal cycler (Applied Biosystems, Foster City, CA) by an initial denaturation step hold at 95 °C for 3 min followed by 45 cycles at 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30s. Signal was captured at each cycle at the end of elongation steps. Melting curve analysis was performed at the end of each run for 95 °C for 15s, 60 °C for 1 minute, 95 °C for 15s and 60 °C for 15s. For each real time PCR run, a Non-Template Control (NTC) was used. The NTC produced negative result for each run showing there was no external contamination. A Positive Amplification Control was used in each run as well. A sample is considered positive for a particular pathogen when an amplification curve is observed (Ct value obtained). Each sample was tested for B. burgdorferi, A. phagocytophilum, B. microti, and B. miyamoti, see Table S1 for each sample’s pathogen Ct scores.