BIOL478/S20:SARS-CoV-2 Testing Exercise
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This assignment is due at the beginning of class on Wednesday, April 15. E-mail a Word document/PDF to Dr. Dahlquist.
Overview
- Read this article: Michael J. Loeffelholz & Yi-Wei Tang (2020) Laboratory Diagnosis of Emerging Human Coronavirus Infections — The State of the Art, Emerging Microbes & Infections, DOI: 10.1080/22221751.2020.1745095
- It has a good overview of how samples are collected and what tests were available in February, when the article was submitted.
PCR Test
This portion of the assignment is based on an actual protocol posted by the Andersen lab at the Scripps Research Institute.
- Review this protocol, and think about how you would need to write this up in your notebook as a pre-lab if we were actually going to do this protocol.
- Protocol for COVID-19 screening using CDC and WHO primers, Version 1.0 (March 18, 2020), by Catie Anderson and Mark Zeller of the Scripps Research Institute
- Watch this video that gives an overview of how qPCR works. The PCR that we did in lab is known as "endpoint" PCR.
- Designing primers
- Check that the primers in the protocol actually bind to the SARS-CoV-2 sequence using BLAST
- For each of the primers listed in the protocol, BLAST them against the Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1, complete genome, GenBank: MN908947.3
- Paste screenshots of your results into your Word document/Google Doc.
- Check the physical-chemical properties of the primers with OligoCalc
- For each of the primers listed in the protocol, determine their properties with the OligoCalc tool.
- Paste screenshots of your results into your Word document/Google Doc.
- Are the annealing temperatures for the qPCR assays compatible with the Tm's of the primers?
- Check that the primers in the protocol actually bind to the SARS-CoV-2 sequence using BLAST
- Optimizing the assay
- This Virtual PCR Simulator allows you to simulate the effects of changing the PCR conditions on the results of the reaction.
- Run the simulation at least 10 times with different conditions to see if you can maximize the yield and purity of your amplified product. If you click on the "trajectory" option in the results, it will show you something that is a little like a qPCR amplification curve.
- Paste screenshots of your results into your Word document/Google Doc. What conditions optimized your results?
- This Virtual PCR Simulator allows you to simulate the effects of changing the PCR conditions on the results of the reaction.
Serum Antibody Test
- Read the press release from Stanford University School of Medicine: Stanford Medicine Develops Antibody Test for Coronavirus April 10, 2020
- Complete the exercise at HHMI Biointeractive Virtual Immunology Lab
- Answer the questions on the worksheet posted on the Google Doc
- Read this article: Amanat, F., Nguyen, T., Chromikova, V., Strohmeier, S., Stadlbauer, D., Javier, A., ... & Caplivski, D. (2020). A serological assay to detect SARS-CoV-2 seroconversion in humans. medRxiv doi: 10.1101/2020.03.17.20037713 and answer the question on the Google Doc.
