Maxwell rsc mirna plasma and serum kit

Viral RNA was extracted from culture supernatant using Maxwell RSC miRNA Plasma and Serum Kit (Promega, Madison, WI, USA), which included a DNaseI digestion step to have only RNA present. SGA of 3′ half genome sequences was performed as previously described.⁶⁵ (link) Complementary DNA was synthesized using the SuperScript IV First-strand Synthesis System (Invitrogen, Carlsbad, CA) with primer R-9632 5′-ACTACTTGAAGCACTCAAGGCAAGCTTTATTG-3′. cDNA was serially diluted with the goal of less than 30% positive amplification by nested PCR using Herculase II Fusion DNA Polymerase (Agilent, California, USA). At this dilution, most positive wells contain amplicons derived from a single cDNA molecule. First round PCR included sense primer F-4875 5′-CAAATTAYAAAAATTCAAAATTTTCGGGTTTATTACAG-3′ and antisense primer R-9626 5′- TGAAGCACTCAAGGCAAGCTTTATTGAGGC-3′. Following PCR conditions were used: 94°C for 2 min followed by 35 cycles of 94°C 15 s, 58°C 30 s, 68°C 4 min, with a final extension of 68°C for 10 min. The second PCR included sense primer F-4900 5′-GGGTTTATTACAGGGACAGCAGAG-3′ and antisense primer R-9602 5′-TGAGGCTTAAGCAGTGGGTTCC-3′. PCR conditions were used: 94°C for 2min followed by 45 cycles of 94°C 15 s, 58°C 30 s, 68°C 4 min, with a final extension of 68°C for 10 min. All PCR products are verified on a 1% agarose gel and sent directly for sequencing.

RNA was extracted from respiratory samples using the Maxwell RSC miRNA Plasma and Serum kit (Promega, Madison, WI, USA). Quantification cycle (Cq) values (i.e., viral RNA loads) were measured by RT-qPCR using the QuantiTect Probe RT-PCR Kit (Qiagen, Hilden, Germany) targeting the SARS-CoV-2 nucleoprotein (N) region via an NIID-N2 primer/probe set.⁴⁶ (link) The thermal cycling conditions were as follows: 50°C for 30 min; 95°C for 15 min; and 45 cycles of 95°C for 15 s and 60°C for 1 min. The Cq values of samples judged to be negative were analyzed by substituting a Cq value of 45. Cq values were converted to viral nucleoprotein RNA copy numbers/reaction according to a previously reported simple regression line.⁴⁷ (link) Viral isolation and PCR screening for mutations of interest (N501Y, E484K, L452R) were attempted for NIID-N2 primer/probe set positive samples.