Figure 2

RT-PCR Analysis of cell line RNA for detection of the alphanodavirus, TNCLV. To determine the sensitivity of TNCLV RNA detection, a segment of TNCLV RNA 1 (2368-2933 nt) was synthesized in vitro and used to spike RNA from Sf9 cells. 250 ng of the in vitro RNA fragment was used for a series of serial dilutions (10^-1 to 10^-12) and each diluted RNA fragment sample was added to 250 ng of Sf9 total cell RNA (representing approximately 3000 cells). One-step RT-PCR was performed using the Invitrogen SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase under the following conditions: 30 min at 45°C, and 94°C for 2 min for 1 cycle; followed by 94°C for 15 sec, 55°C for 30 sec, and 72°C for 45 sec for 40 cycles; then 10 min at 72°C. The sensitivity of detection was estimated as approximately 1 TNCLV RNA per 50 cells. Ao38, High Five and Sf9 cell RNAs were examined for TNCLV using the same primers and conditions for RT-PCR as described for spiking experiments. For High Five cells, total cellular RNA (250 ng; representing an estimated 3000 cells) was diluted as indicated (10^-3 to 10^-5). For analysis of Ao38 and Sf9 cells, 250 ng of cell RNA was analyzed directly by RT-PCR as described above.