Sars cov 2 ddpcr kit

Simultaneous quantification of 2 SARS-CoV-2 nucleocapsid (N) target sequences (N1 and N2) and human RNase P-encoding (RPP30) sequence was performed using the Bio-Rad SARS-CoV-2 ddPCR Kit with the QX200 AutoDG Droplet Digital PCR System (QX200 AutoDG System; Bio-Rad Laboratories, Hercules, CA). Reaction mixtures of 22 µL each were prepared as follows: 5.5 µL of 4x One Step-RT-ddPCR Supermix, 2.2 µL of reverse transcriptase, 1.1 µL of dithiothreitol, 1.1 µL of the 20 × 2019-nCoV CDC ddPCR triplex probe TaqMan assay, and 12 uL of TNA. Of this 22-uL reacture mixture, 20 uL were loaded onto the instrument for testing. Final concentrations of primers and probes were 900 and 250 nmol/L, respectively. The PCR plates were placed on the QX200 AutoDG, followed by RT-ddPCR amplification as follows: 25 °C for 3 min and 50 °C for 60 min, followed by the PCR steps: 95 °C for 10 min, then 40 cycles of denaturation at 95 °C for 30 s and annealing/extension at 55 °C for 1 min, and a final enzyme deactivation at 98 °C for 10 min. Targets in the droplets were counted by the QX200 Droplet Reader, and signal data were analyzed using QuantaSoft Analysis Pro-software version 1.0.596 (Bio-Rad Laboratories, Inc., Hercules, CA). Preparation of amplification plate, amplification, detection, and data analysis required 4.5 hr and 7.5 hr for 24- and 96-sample assay runs, respectively.

After extracted RNA samples were tested for RT-qPCR, they were stored at -80°C until ddPCR testing. The assay uses the QX200 AutoDG Droplet Digital PCR System (Bio-Rad Laboratories, Hercules, CA) following the Bio-Rad SARS-CoV-2 ddPCR Kit EUA Instructions for Use. Viral load measured in RNA extract was corrected for input and output extraction volumes and 10-fold dilution into assay-specific extraction buffer to determine viral load in residual samples applied to Ag RDT as described previously [22] (link).