Nuclisense easymag

Viral load kinetics and duration of viral shedding of SARS-CoV-2 RNA in bodily specimens of all origins (respiratory, faeces, vaginal swab, urine, semen) were determined using an in-house protocol (in-house qPCR) [49 (link)]. Nucleic acids were extracted from 190μl of sample material, in addition to 10μl of internal control (phocine distemper virus (PDV)), using the NucliSense EasyMag or eMAG with the Specific A protocol (bioMerieux, Lyon, France), according to the manufacturer’s instructions. The in-house qPCR targets the SARS-CoV-2 E gene following the description by Corman et al. [50 ] with minor modifications. Analysis is performed using a middleware software referred to as FlowG (LabHelp Labautomation), which can be used for interpretation of the results and communicating them back to the laboratory information system. Samples with a cycle time value (Ct) lower than 34 are considered positive while a Ct of 34-39 is considered inconclusive and the sample is repeated, which is routine clinical practice in our hospital for any new cases. Finally, Ct values above 40 are considered negative. The protocol was adapted to target new SARS-CoV-2 viral variants.

The extraction of viral RNA for both method A and B was performed using NucliSense EasyMag (bioMérieux, Marcy l’Etoile, France): after the incubation for the lysis phase (20 min), magnetic silica was added, and several washes were performed to remove sample residues. A final elution (100 μL) was carried out with TE buffer at pH 8.0. After nucleic acid extraction, the OneStep PCR Inhibitor Removal Kits (Zymo Research, Irvine, CA, USA) was used to remove PCR inhibitors. RT-qPCR SARS-CoV-2 protocol was a one-step viral RNA reverse transcription reaction followed by amplification using a previously published primer/probe set designed in the orf1b, nsp14 region of SARS-CoV2 genome [20 (link)] (Table 1). The standard curve for viral genomic copies (GC) calculation was obtained using serial dilutions of synthetic dsDNA (from 10¹ to 10⁵ GC/µL). Samples negative for the presence of the SARS-CoV-2 viral genome were considered equal to the half of the limit of detection (LOD = 5.8 GC/reaction) obtained by serial dilutions of standard dsDNA. The LOD was the concentration at which over 50% of the technical replicates were positive.

A RT-qPCR respiratory screen previously implemented at the UMCG is routinely used to screen and provide Ct values for 16 respiratory targets: Influenza A virus, Influenza B virus, parainfluenza virus 1-4, human rhinovirus/enterovirus, coronaviruses OC43, NL63, 229E, HKU1, respiratory syncytial virus A and B, adenovirus, bocavirus, and human metapneumovirus. Briefly, nucleic acids were extracted from 190 μL of sample using the NucliSense EasyMag (BioMérieux) and eluted in 110 μL. PDV severed as an IC. A multiplex RT-qPCR using the TaqMan Fast Virus 1-Step kit (ThermoFisher Scientific) was performed using 10 μL of each extracted viral RNA in a total reaction volume of 25 μL (Supplementary Table 3). Amplification was performed on an ABI7500 (Life Technologies) with the following PCR cycling conditions: 2 min 50°C, 20 s 95°C, followed by 45 cycles of 3 s 95°C and 32 s at 60°C. To differentiate between a rhinovirus and enterovirus following the BioFire RP2.0 or respiratory screen, a RT-qPCR targeting the 5’NTR region was performed (see Supplementary Methods).

Stool specimens were washed with phosphate-buffered saline (PBS), and DNA was extracted as described previously (6 (link)). Briefly, 1 mL of stool was washed with PBS, heated for 15 min at 75°C, and homogenized in a bead beater before removing the supernatant for DNA extraction using the NucliSense easyMag automated platform (bioMérieux, Durham, NC). Purified DNA was extracted in a volume of 100 μL.