Nested PCR.

Two nested PCR reactions were performed following reverse transcription-PCR. The first nested PCR reaction amplified the entire eluted RT-PCR product in a 100-μL total volume using Taq polymerase (New England Biolabs), 0.2 μM C-region primers, and 1.55 μM blocking oligos (Table S3), and the following cycling conditions were used: 95 °C for 30 s; 30 cycles of 95 °C for 30 s, 52 °C for 30 s, and 68 °C for 1 min; and one cycle of 68 °C for 5 min. The second nested PCR reaction was performed using LA Taq (New England Biolabs) and 2 μL of the first nested PCR and 0.4 μM primers (Table S3) in a 50-μL total volume with the following cycling conditions: 94 °C for 30 s; 20 cycles of 94 °C for 30 s, 53 °C for 30 s, and 65 °C for 1 min; and one cycle of 65 °C for 5 min. The resulting products were gel-purified and quantified using a QuBit Fluorometer (Life Technologies).