Lightcycler 2.0 thermocycler

The set of 7 primers and TaqMan probe were used for detection of the ntnh gene according to Raphael and Andreadis 2007 [34 (link)]. The reaction was conducted with subsequent concentrations of reagents: 5 μL of DNA, 4 μL of LightCycler TaqMan Master (Roche, Basel, Switzerland), 0.7 μM of each primer and 0.24 μM of NTNH410 TaqMan probe. The real-time PCR was performed using a LightCycler 2.0 thermocycler (Roche, Basel, Switzerland) on the following thermal cycling profile: 10 min at 95 °C as initial denaturation and 40 cycles of denaturation at 95 °C for 15 s, annealing at 42 °C for 15 s, and elongation at 55 °C for 1 min. Validation results of this method for food samples are described in a previous publication [33 ].

The procedures for RNA extraction and real time RT-PCR were followed with minor modifications as described in the OIE terrestrial manual [14] . Two hundred and fifty ml of either OPF or of an anticoagulated blood sample were mixed with 1000 µl Trizol® and RNA was extracted following the instruction of the manufacturer. Viral RNA in samples was reverse transcribed using random hexamers and then quantified by real-time RT-PCR using primers targeting the 3D polymerase region [15] (link). The reaction was performed in a Roche Light Cycler 2.0 thermocycler as previously described [16] (link). Samples that presented a geometric increase in fluorescence emission in two successive cycles prior to cycle number 40 were scored positive, and the first of the two cycles showing emission elevation was considered to be the first cycle of positivity (CP). To convert cycle threshold values generated by real time RT-PCR from experimental samples to RNA genome copies per milligram, serial 10-fold dilutions of plasmid I38 containing the 3D sequence of FMDV (kindly provided by Dr. Soledad Nuñez, Institute of Biotechnology, INTA-Castelar) were analyzed as a quantitative positive control. The number of moles of RNA were calculated as = CP × -0.25 + 11.68.