Quantitative Real-Time PCR of B19V

Quantitative real-time PCR (qPCR) and RT-PCR (qRT-PCR) were performed using the QuantiTect SybrGreen PCR Kit and QuantiTect SybrGreen RT-PCR Kit on a RotorQ system (Qiagen, Hilden, Germany), including 0.5 µM of each specific primer pair, according to previously established protocols [10 (link)]. Quantitation of viral nucleic acids was obtained by the absolute quantitation algorithm, converting quantification cycle (Cq) values to geq number using external calibration curves obtained from respective standard targets. The primers used for the amplification of B19V DNA and the whole set of viral transcripts were the pair R2210–R2355, located in the central exon of the B19V genome, while determination of NS transcripts was obtained by using the pair R1882–R2033. For control and normalization with respect to the number of cells, a target sequence in the region of genomic DNA coding for 18S rRNA (rDNA) was amplified. All primers used are listed in Table 1.