Allplex 2019 ncov assay

NT swabs from all suspected COVID-19 patients underwent rRT-PCR testing for SARS-CoV-2 at the TRC-EID-HSC or at the microbiology laboratory of the Faculty of Medicine, Chulalongkorn University. At TRC-EID-HSC Nucleic acid (NA) extraction was performed on all samples using magLEAD kit according to the manufacturer’s instructions (Precision System Science) and the RT-qPCR was performed using Seegene—Allplex 2019-nCov Assay, in a Bio-Rad CFX 96 qPCR machine according to manufacturer’s instruction. Briefly, reaction was heated to 50oC for 20 minutes for reverse transcription, denatured in 95oC for 15 minutes and then 45 cycles of amplification was carried in 94oC for 15 seconds and 58oC for 30 seconds. Fluorescence was measured using four fluorescence channels: FAM (E gene), HEX (internal control), Cal Red 610 (RdRp gene), Quasar 670 (N gene). The protocol’s stated limit of detection of ORF1ab rRT-PCR was 1000 copies/mL and the cut-off PCR cycle threshold (Ct) was 40.
At the microbiology laboratory the cobas® 6800 system (Roche Diagnostics, Switzerland) was used for detection of SARS-CoV-2 RNA which targeting conserved regions within the ORF 1a/b and E genes, according to manufacturer’s instruction.

H1299-E3 cells were seeded at 1 × 10⁶ cells in 20 ml growth medium in a Corning T75 flask 18–20 h pre-infection. Cells were infected with 10 focus forming units of either D614G, D6, or D190. A 1.5 ml of supernatant was collected at input (day 0) and on days 1–3 post-infection with and culture replenished with1.5 ml fresh medium. Samples were sent to an accredited diagnostic laboratory (Molecular Diagnostic Services, Durban, South Africa) to determine SARS-CoV-2 cycle threshold (Ct) values where samples were extracted using a guanidine isothiocyanate/magnetic bead-based method with the NucliSense (Biomerieux) extractor of the KingFisher Flex 96 (Thermo Fisher). RT-qPCR was performed using the Seegene Allplex 2019 nCoV assay with the Bio-Rad CFX96 real-time PCR instrument as per the kit instructions. RNase P was used as the internal housekeeping gene to monitor extraction and assay efficiency. The kit targets the E, N, and R genes of SARS-CoV-2. Run calls and interpretation was performed by the Seegene Viewer software. Fold-change was calculated as FC = 2^{((mean(Ct  input)—Ct  sample)}.

Concurrent with sample preparation, a second dedicated QIAgility instrument was used for Allplex™ 2019-nCoV assay master mix preparation and aliquoting into appropriate 8-well PCR strips (Bio-Rad Laboratories, USA). Following master mix preparation, the PCR strips were transferred to the sample-addition QIAgility instrument. The sample input volume and master mix constituents are shown in Table 1.
After sample addition, the PCR strips were sealed and briefly centrifuged before being loaded on a CFX96™ Real-Time PCR Detection System (Bio-Rad Laboratories, USA). The real-time PCR cycling parameters recommended by the Allplex™ 2019-nCoV assay package insert were used unchanged. Real-time data analysis was performed using the 2019-nCoV Viewer for Real time Instruments V3 (Ver 3.18.005.003) software as per the Allplex™ 2019-nCoV assay package insert.
If the internal control (RP-IC) was not detected with a cycle threshold (Ct) value <40 and no SARS-CoV-2 targets were detected, the test was deemed invalid and the primary sample was retested with a decreased sample volume input, 2μl instead of 3μl. The remainder of the protocol was unchanged.