TITLE: COVID-19 SCAN molecular workflow
AUTHORS: Lea Starita

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify">Molecular Detection of SARS-CoV-2 for SCAN the Greater Seattle Coronavirus Assessment Network</div><style>
							  .justify:after {
							    content: "";
							    display:inline-block;
							    width: 100%;
							  }
							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify"></div><style>
							  .justify:after {
							    content: "";
							    display:inline-block;
							    width: 100%;
							  }
							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>The SCAN real-time RT-PCR assay for SARS-CoV-2 contains primer/probe sets that target the Orf1b and S genes, designed against the SARS-CoV-2 genome (GenBank: MN908947.3). The primer/probe sets were analyzed </span><span style = "font-style:italic;">in silico</span><span> for specificity for this specific betacoronavirus by Thermo Fisher. All assays are performed on total nucleic acids extracted from upper respiratory tract swabs, including mid-turbinate and nasopharyngeal (NP) swabs stored in Universal Transport Media. The assay limit of detection of 5.6 (Orf1b) and 12.9 (S) molecules per reaction was determined from serial dilutions of synthetic DNA of the target sequences. These probes reproducibly detected SARS-CoV-2 RNA from cultured virus from USA-WA-1/2020 (BEI resources/ATCC) and from 21 specimens confirmed to be positive by the Washington State Department of health. Specificity was determined by lack of detection of SARS-CoV-2 from specimens that are known to be positive for seasonal coronavirus strains HKU1/NL63 or 229E/OC43 and/or other respiratory viruses. These specimens are further inferred to be negative by the fact that they were obtained in the greater Seattle area prior to the first known reports of SARS-CoV-2 in China.</span></div><style>
							  .justify:after {
							    content: "";
							    display:inline-block;
							    width: 100%;
							  }
							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify"></div><style>
							  .justify:after {
							    content: "";
							    display:inline-block;
							    width: 100%;
							  }
							</style></div></div>

[STEPS]
?. Aliquot samples from the original upper respiratory sample found in Universal Transport Medium and transfer into barcoded matrix tubes (Thermo).
?. Transfer  of sample to the extraction plate and store the remainder of sample in .
200 µl
-80 °C
?. Extract total nucleic acids using the Magna Pure 96 automated extractor using the DNA and Viral NA Small Volume Kit (Roche).
?. Place and hold extracted nucleic acids  for the remainder of steps in the protocol.
on ice
?. Using a 96-well pipettor (Liquidator, Rainin), load  of extracted nucleic acids into Taqman assays in quadruplicate into a 384 well plate containing SARS-CoV-2 and RNaseP RT-PCR reactions master mixes:
5 µl
?. Master mix for S target: load into rows A, C, E, G, I, K, M, O using a Mantis liquid dispenser (Formulatrix) in a dead air hood:Master Mix Component 1:  1x TaqPath 1 step RT-qPCR master mix (A15300)Master Mix Component 2:  1x SARS-CoV-2 assay with a FAM fluor (4332079 assay # APXGVC4)Master Mix Component 3:  RNaseP assay with VIC fluor (A30064)
?. Master mix for Orf1B target: load into rows B, D, F, H, J, L, N, P using a Mantis liquid dispenser (Formulatrix) in a dead air hood:Master Mix Component 1: 1x TaqPath 1 step RT-qPCR master mix (A15300)Master Mix Component 2: 1X SARS-CoV-2 assay with a FAM fluor (4332079 assay # APGZJKF)Master MIx Component 3:  RNaseP assay with VIC fluor (A30064)
?. Load positive control DNA into 2 positive control wells for each target and add water to the 4 no template control wells.
?. Seal plate with a clear adhesive (Bio-Rad MSB1001).
?. Perform RT-PCR on QuantStudio 6 (Applied Biosystems) on the Fast setting according to manufacturer instruction.