Sequence detector version 1

CVB4E2 positive strand RNA was quantitated by two-step quantitative RT-PCR as described previously (25 (link)). Briefly, total RNA was extracted with RNeasy minikit (Qiagen, Valencia, Calif.) and resuspended. Total RNA was measured by a quantitative RT-QPCR for RNA with the Affinityscript QPCR cDNA synthesis kit and the brilliant II QPCR kit (Agilent technologie stratagene). Positive strand specific RT was carried out on extracted RNA by using the reverse primer at 42°C for 15 min. PCR was performed with universal cycle conditions (10 min at 95°C, 40 cycles of 30 s at 60°C) on a Mx3000p (stratagene). The following primers, used to detect CVB4E2 RNA, were located within the enterovirus 5′-nontranslated region, which is highly conserved among enterovirus serotypes: CVB4 forward (5′-CCC TGA ATG GGG CTA ATC) and CVB4 reverse (5′-ATT GTC ACC ATA AGC AGC CA). The sequence of the CVB4 probe was 5′-VIC-AAC CGA CTA CTT TGG GTG TCC GTG TTT-TAMRA (Applied Biosystems). The absence of contaminating DNA in samples was checked by RT-PCR without the reverse transcriptase enzyme. Primers and probe pairs were designed with PrimerExpress software, and the data were analyzed with Sequence Detector version 1.6.3 (both from Perkin-Elmer, Boston, Mass. Results were expressed as cycle threshold (Ct) which is inversely proportionate to RNA level.

CVB4E2-positive strand RNA was quantitated by two-step quantitative RT-PCR as described previously (15 (link)). Briefly, total RNA was extracted with the RNeasy minikit (Qiagen, Valencia, California). Total RNA was retro-transcribed to cDNA using the Affinityscript QPCR cDNA synthesis kit and a specific reverse primer at 42°C for 15 min. Positive strand-specific RT was quantitated by the brilliant II QPCR kit (Agilent Technologies Stratagene) under universal cycle conditions (10 min at 95°C, 40 cycles of 30 s at 60°C) on a Mx3000p (Stratagene). Primers were located within the enterovirus 5′-nontranslated region, which was highly conserved among enterovirus serotypes: CVB4 forward (5′-CCCTGAAT GGGGCTAATC) and CVB4 reverse (5′-ATTGTCACCATA AGCAGCCA). The sequence of the CVB4 probe was 5′-VICAACCGACTACTTTGGGTGTCCGTGTTT- TAMRA (Applied Biosystems). Negative controls were performed by RT-PCR without the reverse transcriptase enzyme or without DNA. Primers and probe pairs were designed with PrimerExpress software, and data were analyzed with Sequence Detector version 1.6.3 (both from Perkin- Elmer, Boston, Massachusetts). Results were expressed as the cycle threshold (Ct)