Quantification of B19V Nucleic Acids

Equal amounts of cell cultures, corresponding to 1.5 × 10⁵ cells, were collected as appropriate at 2 or 48 h post-infection (hpi) and processed by using the Maxwell Viral Total Nucleic Acid kit on a Maxwell MDx platform (Promega), to obtain a total nucleic acid fraction in elution volumes of 150 μL. The quantitative evaluation of target nucleic acids was carried out by qPCR assays in a Rotor-Q system (Qiagen, Hilden, Germany). For the analysis of B19V DNA, aliquots of the eluted nucleic acids (corresponding to ~500 cells) were directly amplified in a qPCR assay (Maxima SYBR Green qPCR Master Mix, Thermo Scientific, Life Technologies, Monza, Italy). For the analysis of B19V RNA, parallel aliquots were first treated with the Turbo DNAfree reagent (Ambion, Life Technologies) before amplification in a qRT-PCR assay (Express One-step SYBR GreenER Kit, Invitrogen, Life Technologies). Standard cycling programs were used, followed by a melting curve analysis to define the $T_{\mathrm{m}}$ of amplified products. The primer pair R2210–R2355, located in the central exon of B19V genome, was used to amplify both viral DNA and total RNA, and a target sequence in the region of genomic DNA coding for 5.8S rRNA (rDNA) was amplified in parallel reactions for normalisation [5 (link),31 (link)].