Nanoplates

The digital PCR (dPCR) procedure was performed following the manufacturer’s instructions using the QIAcuity Four platform (Qiagen). All dPCRs were performed in a total reaction volume of 40 µL for 24-well (26 k partitions) Nanoplates (Qiagen, 250001), using the QIAcuity One-Step Viral RT-PCR Kit (Qiagen, 1123145). All reactions were set up as singleplex containing 0.6 µM forward primer, 0.8 µM reverse primer, and 0.25 µM of target probe with 5 µL of template. Each plate contained a no template control (NTC). Amplification conditions were as follows: 50°C for 40 min for reverse transcription, 95°C for 2 min for enzyme activation, 95°C for 5 s for denaturation, and 60°C for 30 s for annealing/extension for a total of 40 cycles). Imaging was carried out by reading in the Green/FAM channel.
Data acquisition and analysis were carried out using the QIAcuity software version 2.0.20. Thresholds were set based on the amplitude signal observed in negative control samples (19 (link), 20 (link)). Experiments were performed on three separate occasions as duplicate replicates per experiment. The same threshold was applied to all wells per target of one PCR plate. The obtained quantity data with a confidence value range of 1.7%–6.7% and lambda range of 0.035–2.336 were included in the final quantitation calculations.

SARS CoV-2 RNA was detected and quantified in 5 μL of total RNA obtained from stool and urine specimens using the SARS CoV-2 N1 + N2 assay kit according to manufacturer’s instructions on a QX-200 ddPCR platform (Qiacuity QX-200, Qiagen, Germany) and a recent published literature on waste water (11 (link)). The SARS CoV-2 CoV-2 N1 + N2 Assay is a mixture of four primers and two probes purified by HPLC at a 20x concentration. These four primers are based on the CDC design, targeting the regions N1 and N2 of the viral genome. The two probes are coupled with FAM as a reporter dye and use ZEN^™ quenchers for enhanced sensitivity. For the N1 and N2 assays, the concentrations of the primer and probe, as well as the annealing temperature and duration, were optimized. N1 and N2 assays were carried out in 40 μL reaction mixtures using the QIAcuity One-Step Viral qRT-PCR Kit (Cat no. 1123145, Qiagen) on 26,000 24-well Nanoplates under ideal circumstances (catalog no. 250001, Qiagen). The microfluidic dPCR plates 26,000 QIAcuity 24-well Nanoplates enable 24 samples to be run with up to 26,000 partitions/well. Each partition has a volume of 0.91 nL and the PCR takes place within each partition.