Viral Identification
NPS were processed by the lab and their extraction was processed using
the automated MagNA Pure system (Roche, Basel, Switzerland). The
majority of samples were tested with the LightMix® Modular SARS and
Wuhan CoV E-gene kit (targeting the E-gene; TIB Molbiol, Berlin,
Germany) for the SARS-CoV-2 PCR. Some were tested using the
AusDiagnostics Respiratory Pathogens 16-well assay (Mascot, Australia),
on the AusDiagnostics High-Plex 24 system (the SARS-CoV-2 target of this
assay is the ORF-1 gene). Respiratory panel testing was by Roche
LightCycler 480 Instrument II viral panel30.
Viral RNA was manually extracted from 140 μL of NPS, saliva and 140 μL
of 20% (w/v) faecal suspension 31 and then eluted in
60 μL sterile, molecular grade water (Life Technologies, Australia),
using the QIAamp viral RNA kit (QIAGEN, Hilden, Germany) according to
the manufacturer’s instructions. The Centers for Disease Control and
Prevention (CDC) developed a real-time reverse transcription PCR panel
targeting nucleocapsid protein genes, N1 and N232.
CDC’s validated platform was selected for saliva and stool analyses.
SARS-CoV-2 standard (Exact Diagnostic, USA) was used as the standard
curve in each assay to determine viral load.
Whole genome sequencing was conducted on a subset of 12 participants
from 7 households. Briefly, viral RNA from saliva or stool (extracted as
described above) was amplified using the ARTIC version 3 primers and
published protocols and subjected to Illumina sequencing as previously
described33. Following quality trimming, reads were
aligned to the reference genome (Wuhan Hu-1; GenBank MN908947.3) and
consensus sequences generated utilising Geneious Prime. Samples were
classified into the recognised SARS-CoV-2 lineages using
Pangolin34.