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

In each run, the ACH-2 standard curve (using primers that bind the R and U5 regions of the HIV-1 LTR) was used to determine experimentally the provirus content of a dilution series of nonpreamplified IS (Fig. 4A) . These numbers were then assigned to the Alu-gag signals arising from a matching dilution series of preamplified IS (Fig. 4B) . The first PCR serves as a preamplification of Alu-gag signals, reducing the threshold cycle number of preamplified IS in the kinetic PCR. This shifted the IS curves to the left in Fig. 4B by about 12 cycles, corresponding to roughly 4 logs of amplification. Observed amplification factors (average of 6 performed within one assay) are listed in Table 1 . In this particular assay, as few as 5 proviruses in a 50-l sample were detectable. Since each sample can contain up to 50,000 cells, 1 provirus in 10,000 cells could be reliably measured in this run. This sensitivity should be sufficient to measure integration in primary lymphocytes (7, 29) . The relative uncertainty increases with increasing dilution, likely due to accumulated sampling errors. Within a run, the assay is very reproducible: duplicate reactions on the same plate containing the same master mix typically overlay each other closely (Fig. 4B) . Compared to the ACH-2 standard (Fig. 3A) , the dynamic range of the preamplified IS is relatively narrow, usually about 2.5 logs. These limits are attributable to the sigmoidal amplification characteristics of the nonkinetic preamplification step (13, 32) . Within this range, however, the standard curve is very linear (Fig. 4C) . The y intercept for this regression line typically corresponds to threshold cycles in the mid-twenties, with a slope of roughly Ϫ2.5 cycles per 10-fold increase in analyte concentration.