Rox dye 2

For real-time RT-PCR analysis of SARS-CoV-2, the N1 assay was used for method development and both N1 and N2 assays (Lu et al., 2020 (link)) were used for the final RV-RT-PCR method evaluation with spiked swabs. The primers and probes were supplied by Biosearch Technologies (Novato, CA; Cat. No. KIT-NCOV-PP1−1000). Ten-fold dilutions of a synthetic RNA standard (BEI Resources; Cat. No. NR-52358), resulting in levels ranging from 1.5 to 1.5 × 10⁵ viral genome copies per RT-PCR reaction, were run with each PCR plate along with a negative control (nuclease-free water only).
Each 25-μL RT-PCR reaction contained 12.5-μL 2X Master Mix and 0.5-μL ROX Dye II (One Step PrimeScript™ III RT-PCR Kit; Takara Bio, Mountain View, CA; Cat. No. RR600B), 0.625-μL primers/probe (5 μM probe, 20 μM each forward and reverse primers), 6.375-μL PCR-grade water, and 5-μL RNA extract. An Applied Biosystems 7500 Fast Real-Time PCR Instrument (Thermo Fisher Scientific) was used with the following thermocycling conditions: 50°C for 15 min; 95°C for 2 min; 45 cycles of 95°C for 3 s and 55°C for 30 s.

The integrity of cfDNA was determined by qPCR as previously reported 12 (link). Two primer sets (Sangon Biotech) were used to amplify ALU sequence: the primer set for 115 bp ALU amplicon was: forward 5'-CCTGAGGTCAGGAGTTCGAG-3' and reverse 5'-CCCGAGTAGCTGG GATTACA-3'; the primer set for 247 bp ALU amplicon was: forward 5'-GTGGCTCACGCCTGTAATC-3' and reverse 5'-CAGGCTGGAGTGCAGTGG-3'. The qPCR was done in a final volume of 20 μL on the ABI 7500 (Applied Biosystems) in triplicate and each reaction mixture contained 2 μL cfDNA template, 0.5 μL of each forward and reverse primer (10 μM, ALU115 or ALU247), 10 μL 2x SYBR Green (Takara), 0.5 μL Rox Dye II, and 6.5 μL RNase-free water. The reaction condition was 95 ^oC for 10 s, followed by 40 cycles of 95 ^oC for 5 s, and annealing at 60 ^oC for 34 s. The 115-bp ALU amplicon represented the total amount of DNA fragments including both short and long copies whereas the 247-bp ALU amplicon only reflected the amount of long DNA fragments. The amount of ALU 115 and ALU 247 DNA fragments was determined by comparing the CT values of each sample against the calibration curve created by performing qPCR with serial diluted cDNA (100 ng to 0.01 pg; EASY Dilution, Takara). cfDNA integrity was calculated as the relation of ALU 247 to ALU 115 according to the methods of Umetani et al 12 (link).