Qiacuity qx 200

Using a CFX96^™ thermocycler and the Qiagen SARS CoV-2 N1 + N2 assay kit (Cat. No. 222015, Qiagen, Germany), qRT-PCR was carried out with 5 μL of total RNA isolated from stool and urine samples (Qiacuity QX-200, Qiagen, Germany). The N1 and N2 genes, which code for the viral nucleocapsid, the E gene, which codes for the viral envelop, as well as the RNAse P gene as an internal control, are all detected by this kit. In accordance with the manufacturer’s recommendations, samples were deemed positive for SARS CoV-2 if any of the genes (E or N) it detects had a Ct value below 37.
All the twenty normal samples showed negative results by using Q-PCR targeting this multiplex N1 + N2 assay kit.

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.