Determination of daily reproduction numbers of SARS-CoV2 based on death cases suggests more rapid initial spread in Italy and the United States

Armin Ensser; Klaus Ueberla

Southern blotting. After G418 selection, total DNA was prepared as described elsewhere (DNeasy tissue system; Qiagen) from the IS, from uninfected CEM-SS cells, and from CEM-SS cells that had been acutely infected with HIV-1 IIIB (300 ng of p24 Gag /ml) by spinoculation 20 h prior to harvest. After electrophoresis in a 0.8% agarose gel (loading 20 g of DNA per lane), the DNA was transferred to a charged nylon membrane (GeneScreen Plus; Perkin-Elmer). The membrane was prehybridized with PerfectHyb PLUS hybridization buffer (Sigma) and then probed with an [␣-32 P]dCTP-labeled probe generated by random priming (RadPrime kit; Invitrogen) the entire HIV-1 IIIB genome. After washing, the blot was exposed to BioMax MR film (Kodak) in a BioMax TranScreen LE intensifying screen (Kodak) for 1 week at Ϫ80°C. Two-step PCR amplification. The initial nonkinetic preamplification was performed on dilutions of the IS cells as well as dilutions of unknowns. It is essential to preamplify a series of dilutions of both the standards and unknowns to insure that PCR substrates are not limiting. Reactions in which these reagents are limiting lack a dose response compared to other dilutions in the standard curve. The sequences of the preamplification primers were as follows: genomic Alu forward, 5Ј-GCC TCC CAA AGT GCT GGG ATT ACA G-3Ј; and HIV-1 gag reverse, 5Ј-GCT CTC GCA CCC ATC TCT CTC C-3Ј. The reaction conditions were 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl 2 , 1 mM concentration of mixed dNTPs, 50 mM KCl, 100 nM Alu forward primer, 600 nM gag reverse primer, and 0.05 U of Platinum Taq DNA polymerase (Invitrogen Life Technologies). The thermal cycler (DNA Engine PTC-200; MJ Research) was programmed to perform a 2-min hot start at 94°C, followed by 20 steps of the following: denaturation at 93°C for 0.5 min, annealing at 50°C for 1 min, and extension at 70°C for 1 min 40 s. Linear, one-way amplification was also monitored by performing the preamplification PCR with either the gag primer alone or the Alu primer alone.