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

The second-round real-time quantitative PCR was performed using 25 l of the material from the preamplification or matched dilutions of both the nonpreamplified IS and the nonpreamplified unknowns (acutely infected cells in the presence and absence of integrase inhibitors). These were run with an HIV-1 copy number standard prepared from graded doses of ACH-2 cells. The sequences of the primers were as follows: LTR forward, 5Ј-GCC TCA ATA AAG CTT GCC TTG A-3Ј; and LTR reverse, 5Ј-TCC ACA CTG ACT AAA AGG GTC TGA-3Ј. The LTR molecular beacon probe, labeled at its 5Ј terminus with the reporter fluorophore 6-carboxyfluorescein (FAM) and at its 3Ј terminus with the quencher 4-(4Ј-dimethylamino-phenylazo)-benzene (DABCYL), had the following sequence: 5Ј-FAM-GCG AGT GCC CGT CTG TTG TGT GAC TCT To express integration as a ratio of proviruses per target cell, a kinetic PCR assay for ␤-globin DNA was used to determine cell numbers on the same plates used for HIV-1 quantitation. A standard curve for cellular DNA was prepared by serially diluting a CEM-SS lysate starting at 4 ϫ 10 6 /ml (prepared as described above). The sequences of the ␤-globin forward and reverse primers were 5Ј-CC CTTGGACCCAGAGGTTCT-3Ј and CGAGCACTTTCTTGCCATGA-3Ј, respectively. The ␤-globin molecular beacon sequence was 5Ј-FAM-GCGAGCA TCTGTCCACTCCTGATGCTGTTATGGGCGCTCGC-DABCYL-3Ј. React ions were carried out in a volume of 50 l containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 3.5 mM MgCl 2 , 500 nM ROX, 0.8 mM concentration of mixed dNTPs, 1 M concentration of forward and reverse ␤-globin primers, 100 nM molecular beacon probe, and 0.025 U of Platinum Taq DNA polymerase. The thermal programs were described above.