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

Sensitivity and specificity of the assay. To determine whether linear, one-way amplification of unintegrated HIV-1 (Fig. 5A) . This construct lacks an Alu repeat, so when either the gag primer alone or the Alu-gag primer pair was used in the preamplification, similar signals were obtained, corresponding to roughly a fivefold amplification. As expected, when the Alu primer was used alone in the preamplification of pIIIB, a signal was obtained which was comparable to that of a nonpreamplified plasmid sample. Signals corresponding to up to 10 4 copies of unintegrated DNA can be tolerated by this assay, since they appear at higher threshold cycle numbers than the most dilute of the IS-derived preamplified standard curves. The sensitivity of the assay was further examined by performing one-way and bidirectional preamplifications of the IS, a heterogeneous template that contains exclusively integrated DNA (Fig. 5B) . Again, one-way amplification contributed a low-level interfering signal corresponding to about fivefold amplification over the nonpreamplified standard. In contrast, bidirectional Alu-gag preamplification resulted in a 4 log net amplification (approximately a 12-cycle reduction in threshold cycle number). Taken together, these findings indicate that the interfering signal from one-way amplification is small and can be subtracted from the total signal obtained from any given sample. Accounting for this signal becomes important at early time points following acute in vitro HIV-1 infection, when proviruses comprise only a minority of the total HIV-1 DNA present.