COVID-19 Testing Flashcards
SARS CoV-2
- related to SARS CoV-1
- enveloped
- (+) ssRNA virus
- 60-140 nm
- 29.9 kb with 14 ORFs
transmission of coronavirus
respiratory droplets
the national case definition of COVID-19 required at ____ gene targets to be detected
2
E gene assay
more sensitive, cross-reacts with SARS-CoV-1
RdRp gene assay
less sensitive but more specific
easyMag most suitablee use
lower resp samples (viscous(
this is most suitable everything else
MagMax
Hamilton Starlet
too slow so tried for sampling pooling but takes 3 hours; doesnt work for workflow of doing thousands of tests a day
variant of concern
demonstrated to be associated with increased transmissibility or virulence, or reduced effectiveness ff public health measurs, available diagnostics, vaccines, or therapeutics
variant of interest
has genetic changes predicted or known to affect viral characteristics, and identified as cause of insignificant community transmission or multiple disorders
most sensitive tests for respiratory viruses
molecular testing
T or F. pan coronavirus RT-PCR is gel-based.
T
- obtained primers for a gel-based SARS-CoV-2 specific RT-PCR based on the RdRp gene sequence and performed a quick evaluation
how to test for analytical specificity?
pathogens that can be encountered in pt samples where illness may be similar were tested by both E and RdRp gene assays
- cross rxn only observed in E gene assay for the original SARS-CoV (as expected)
When the E and RdRp gene assays were implemented, what was done to confirm test performance?
first 100 neg samples and 10 pos samples confirmed by sending to NML
initial covid-19 test
Pan-coronavirus RT-PCR (gel-based)
obtained primers for a gel-based SARS-CoV-2 specific RT-PCR based on the RdRp gene sequence and performed a quick evaluation
advantages of real time PCR
easy to interpret, run, automate, and scale up to high test volumes
considerations:
- analytical sensitivity (limit of detection)
- analytical specificity (cross-reactivity)
- accuracy
- precision
- where to find positive and negative samples???
- what test to use to compare results (ie, gold std?)
analytical sensitivity test
known conctns of in-vitro RNA were each tested nine times and probit (pos/neg) analysis was performed
multiplex assays
created in March to increase capacity (before, could only run 32 samples on a 96-well plate)
- E, RdRp and internal control all in one well, all 96 wells could be used
multiplex challenges
- RdRp gene with high background and an early CT value
- E gene with high background and a late CT value
why did the gene target requirement change to just one?
COVID-19 less rare = became less important to confirm ever sample with two gene targets
- wanted to get rid of high background
- replaced FAM dye with NED dye (less intense so less background and less false pos)
quadplex assay
fall 2021; influenza szn
- introduce influenza testing while maintaining COVID-19 diagnostics
- four targets in a single PCR reaction = SARS-CoV-2, influenza A, influenza B, internal control
