This document summarizes a class on detecting, quantifying, and tracking variations of SARS-CoV-2 RNA from COVID-19 samples. It discusses using quantitative RT-PCR (qRT-PCR) to detect and measure viral RNA levels in samples. Sequencing is used to identify variations in the viral genome over time, and online tools like Nextstrain allow viewing the evolution and global transmission of variants. Genotyping assays are also described that can rapidly screen samples for known single nucleotide variations during PCR.

1. EVE 198-COVID-19 (Mass) Testing
COVID-19 Mass Testing Course (EVE198, SS21)
Class 4 - SARS-CoV-2 RNA Detection, Quantification and Variation

2. EVE 198-COVID-19 (Mass) Testing
SARS-CoV-2 RNA Detection, Quantification and Variation
• Detection and Quantification
• Sequencing
• Variants & Evolution
• Genotyping

3. EVE 198-COVID-19 (Mass) Testing
SARS-CoV-2 RNA Detection, Quantification and Variation
• Detection and Quantification
• Sequencing
• Variants & Evolution
• Genotyping

4. EVE 198-COVID-19 (Mass) Testing

5. EVE 198-COVID-19 (Mass) Testing

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Detection and Quantification
• Collect samples
• Store samples
• Lyse virus
• Extract RNA (sometimes skipped)
• q RT PCR

7. EVE 198-COVID-19 (Mass) Testing
qRT PCR Details
• q = quantitative
• RT = real time
• RT = reverse transcriptase
• (sometimes call real time reverse transcriptase PCR)
• PCR = polymerase chain reaction

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qRT PCR Details
Baselineis defined as PCR cycles in which a
reporter fluorescent signal is accumulating but is
beneath the limits of detection of the instrument.
ΔRnis an increment of fluorescent signal at each
time point. The ΔRn values are plotted versus the
cycle number.
Threshold is an arbitrary level of fluorescence
chosen on the basis of the baseline variability. A
signal that is detected above the threshold is
considered a real signal that can be used to
define the threshold cycle (Ct) for a sample.
Threshold can be adjusted for each experiment so
that it is in the region of exponential amplification
across all plots.
Ct is defined as the fractional PCR cycle number
at which the reporter fluorescence is greater than
the threshold. The Ct is a basic principle of real
time PCR and is an essential component in
producing accurate and reproducible data.

9. EVE 198-COVID-19 (Mass) Testing

10. EVE 198-COVID-19 (Mass) Testing
SARS-CoV-2 RNA Detection, Quantification and Variation
• Detection and Quantification
• Sequencing
• Variants & Evolution
• Genotyping

11. EVE 198-COVID-19 (Mass) Testing

12. EVE 198-COVID-19 (Mass) Testing
SARS-CoV-2 RNA Detection, Quantification and Variation
• Detection and Quantification
• Sequencing
• Variants & Evolution
• Genotyping

13. EVE 198-COVID-19 (Mass) Testing
NY Times Coronavirus Variant Tracker
https://www.nytimes.com/interactive/2021/health/coronavirus-varian
t-tracker.html

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18. EVE 198-COVID-19 (Mass) Testing
For more on the B.1.1.7 mutations, see: Inside the B.1.1.7 Coronavirus Variant.

19. EVE 198-COVID-19 (Mass) Testing
Nextstrain
https://nextstrain.org/sars-cov-2/
https://nextstrain.org/ncov/gisaid/global

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21. EVE 198-COVID-19 (Mass) Testing
Phylogeny

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Distribution

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Variation across
Genome

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Taxa, Clade,
Samples, etc

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Attempt at Live Demo ...
https://nextstrain.org/ncov/gisaid/global

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Attempt at Another Live Demo ...
https://clades.nextstrain.org

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SARS-CoV-2 RNA Detection, Quantification and Variation
• Detection and Quantification
• Sequencing
• Variants & Evolution
• Genotyping

29. EVE 198-COVID-19 (Mass) Testing
Genotyping
• Sequencing identifies key differences between variants
• Develop assay to screen for variants at single positions in the
genome (e.g., A vs G)
• Can run separately from or simultaneous to qRT-PCR
• Advantages: Fast, Cheap
• Disadvantages: Limited # of sites. Only screen for known variation.

30. EVE 198-COVID-19 (Mass) Testing
Example of genotyping during PCR

31. EVE 198-COVID-19 (Mass) Testing
Questions