Apr 01, 2020
COVID-19 SCAN molecular workflow
- 1University of Washington
Protocol Citation: Lea Starita 2020. COVID-19 SCAN molecular workflow. protocols.io https://dx.doi.org/10.17504/protocols.io.bebkjakw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 27, 2020
Last Modified: April 01, 2020
Protocol Integer ID: 34892
Keywords: COVID-19, RT-PCR, Seattle
Abstract
Molecular Detection of SARS-CoV-2 for SCAN the Greater Seattle Coronavirus Assessment Network
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 in silico 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.
Attachments
Guidelines
Controls for validation:
Positive control 1 for RT-PCR:
Control RNA is from BEI Resources, NR-52285 Genomic RNA from SARS-Related Coronavirus 2, Isolate USA-WA1/2020 (lot# 70033320). LoD will be performed with this RNA.
Positive control 2 for RT-PCR:
Synthesized DNA (Life Technologies A47533) of the RT-PCR target spiked into nucleic acids from upper respiratory samples collected prior to November, 2019. LoD will be performed with this DNA.
Positive control 3 for RT-PCR:
Samples known to contain SARS-CoV-2. These samples were identified by research testing and confirmed to be positive by sequencing and additional testing by the Washington Department of Health.
Internal Control for sample quality and extraction is human RNaseP:
Extraction Control 1:
50,000 cells from human cell line HAP1, these samples are positive for RNaseP by RT-PCR.
Extraction Control 2:
Each respiratory specimen should be positive for human RNaseP by RT-PCR.
Materials
MATERIALS
Nuclease-Free WaterThermo Fisher ScientificCatalog # AM9939
TaqPath™ 1-Step RT-qPCR Master Mix, CGThermo FisherCatalog #A15300
TaqMan™ RNase P Assay, VIC™ dye/QSY™ probeThermo FisherCatalog #A30064
TaqMan™ 2019nCoV Control Kit v1Thermo Fisher ScientificCatalog #A47533
60X custom Orf1B assay with FAM fluor (#APGZJKF)Thermo Fisher ScientificCatalog #4332079
60X custom S assay with FAM fluor (#APXGVC4)Thermo Fisher ScientificCatalog #4332079
MagNA Pure 96 DNA and Viral NA Small Volume KitRocheCatalog #06543588001
Equipment
- MANTIS automated liquid handler (Formulatrix)
- Liquidator (Eppendorf)
- QuantStudio 6 RT-qPCR instrument (Applied Biosystems)
- Dead air hood
Supplies
- MicroB Seal (BioRad MSB1001)
- 384-well plate (Thermo)
- Microfuge tubes
- Pipettors and barrier tips
- Centrifuge with plate adaptor
Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Aliquot samples from the original upper respiratory sample found in Universal Transport Medium and transfer into barcoded matrix tubes (Thermo).
Transfer 200 µL of sample to the extraction plate and store the remainder of sample in -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 On ice for the remainder of steps in the protocol.
Using a 96-well pipettor (Liquidator, Rainin), load 5 µL 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:
Layout of the plate for the assay
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.