COVID-19 Testing

Question 1

SARS CoV-2

Answer 1

• related to SARS CoV-1
• enveloped
• (+) ssRNA virus
• 60-140 nm
• 29.9 kb with 14 ORFs

Question 2

transmission of coronavirus

Answer 2

respiratory droplets

Question 3

the national case definition of COVID-19 required at ____ gene targets to be detected

Answer 3

2

Question 4

E gene assay

Answer 4

more sensitive, cross-reacts with SARS-CoV-1

Question 5

RdRp gene assay

Answer 5

less sensitive but more specific

Question 6

easyMag most suitablee use

Answer 6

lower resp samples (viscous(

Question 7

this is most suitable everything else

Answer 7

MagMax

Question 8

Hamilton Starlet

Answer 8

too slow so tried for sampling pooling but takes 3 hours; doesnt work for workflow of doing thousands of tests a day

Question 9

variant of concern

Answer 9

demonstrated to be associated with increased transmissibility or virulence, or reduced effectiveness ff public health measurs, available diagnostics, vaccines, or therapeutics

Question 10

variant of interest

Answer 10

has genetic changes predicted or known to affect viral characteristics, and identified as cause of insignificant community transmission or multiple disorders

Question 11

most sensitive tests for respiratory viruses

Answer 11

molecular testing

Question 12

T or F. pan coronavirus RT-PCR is gel-based.

Answer 12

T
- obtained primers for a gel-based SARS-CoV-2 specific RT-PCR based on the RdRp gene sequence and performed a quick evaluation

Question 13

how to test for analytical specificity?

Answer 13

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)

Question 14

When the E and RdRp gene assays were implemented, what was done to confirm test performance?

Answer 14

first 100 neg samples and 10 pos samples confirmed by sending to NML

Question 15

initial covid-19 test

Answer 15

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

Question 16

advantages of real time PCR

Answer 16

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?)

Question 17

analytical sensitivity test

Answer 17

known conctns of in-vitro RNA were each tested nine times and probit (pos/neg) analysis was performed

Question 18

multiplex assays

Answer 18

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

Question 19

multiplex challenges

Answer 19

• RdRp gene with high background and an early CT value
• E gene with high background and a late CT value

Question 20

why did the gene target requirement change to just one?

Answer 20

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)

Question 21

quadplex assay

Answer 21

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