Presentation 4: Detection of SARS-CoV-2 from Exhaled Breath

Question 1

What is the current problem in detected SARS-CoV-2?

Answer 1

Current testing methods lack the ability to detect the virus in the lower respiratory tract
Issue because most fatalities occur due to the involvement of the lower respiratory tract

Question 2

What is the gold standard of testing for COVID-19?

Answer 2

RT-PCR

Question 3

How can DNA barcoding be used to detect COVID-19?

Answer 3

1) Assign barcodes to patients
2) RT-PCR
3) Amplicon Sequencing (NGS)
4) Amplicon Matching and Read Counts
5) Results
Same happens when specimens are pooled (NP, OP, Sputum)

Question 4

What is DNA barcoding to detect COVID-19?

Answer 4

Based on current PCR practices coupled with NGS, detect infected individuals when samples are pooled, can predict viral variants

Question 5

What is the Bubbler?

Answer 5

Breath-based detection method
Relies on viral RNA detection
Better indicator of current infection

Question 6

How is the Bubbler set up?

Answer 6

Air vent
Sample 
15 mL Falcon TUbe 
Mineral Oil (Cold) 
RT reaction mix ( pooled primers targeting SARS-COV-2)

Question 7

How does the Bubbler compare to existing technologies?

Answer 7

Less invasive, better measure for risk of contagion as it measures RNA in lungs, can detect SARS- COV-2 in the air, samples airborne viral particles
But…
Risks associated with sputum induction
Contamination of exhaled breath
Does not identify content from cells with capacity to replicate the virus

Question 8

How were the samples collected?

Answer 8

1) Exhaled breath using bubbler

2) 2 tongue scrapes for the control

Question 9

How were the samples prepared for RT-PCR?

Answer 9

Scrapes were dipped in RT reaction mix 
Samples heated to inactivate virus 
RT reaction mixture mixed with:
PCR primers 
SYBR green 
Nuclease free water

Question 10

How was the RT-PCR performed?

Answer 10

1) Hold Stage
2) PCR * 40 cycles
3) Melt Curve
GoTaq master mix was used to detect 18s rRNA and ACE-2 receptor

Question 11

What was determined regarding the ACE-2 receptor?

Answer 11

Tongue was positive for ACE-2, while the bubbler was not

Indicates Bubbler samples breath from lower respiratory tract

Question 12

What was determined about the PCR performed in the bubbler?

Answer 12

Bubbler samples demonstrated RT-PCR efficiency that was similar to RNA extracted from cultured cells, and performing it at the site of collection eliminates RNA extraction and stabilization steps
More rRNA can be detected from the breath than from congenitally extracted RNA

Question 13

What was determined about the Bubbler?

Answer 13

Results from the bubbler are similar to conventional tests for predicting abnormal chest X-Rays
Bubbler is good alternative to sampling lower respiratory tract

Question 14

Why did the researchers perform IVT?

Answer 14

To test using the Bubbler as a control

Developed a high throughput assay to improve detection of SARS-CoV-2 RNA

Question 15

How did the researchers develop a high throughput assay?

Answer 15

Developed a plan to improve detection using IVTof SARS-CoV-2 RNA
Created a barcoded reverse transcription primer:
–> Barcode
–> UMI to distinguish amplicons from random duplication
–> T7 promoter to amplify signal
–> RT primer: targeted SARS-CoV-2 gene RNA or 18s RNA

Question 16

How did the researchers determine number of IVT SARS- CoV-2 gene RNA in each serial dilution?

Answer 16

Correlation between expected barcode reads and observed reads
Detection of the assay was lost at 5th dilution, lowest detection was 334 genomic copies

Question 17

How can the Bubbler be used to detect SARS- CoV-2 in the air?

Answer 17

Add vacuum pump and filed with mineral oil, water, and variety of humidifiers
H1: 79 bp
H2: 69 pb
H3: 59 bp

Question 18

What happened when the researchers used the bubbler to detect SARS- CoV-2 in the air?

Answer 18

Room with high air flow: Upstream bubbler detected all H’s, but downstream bubbler detected only H1 (79 bp)
Room with low air flow: Bubbler captured all Hs
No control group, bad design

Question 19

Why is the bubbler so special compared to an oral scrape or nasal swab

Answer 19

Bubbler: Current infection, Lower Respiratory Tract, Low aerosalization risk, and has environmental applications
The swabs: Current and solved infection, Upper Respiratory tract, have an aerosalization risk, and do not have environmental applications

Question 20

What is RT-LAMP?

Answer 20

Reverse Transcription Loop Mediates Isothermal Amplification
Selecetive amplification of target nucleic acids
Amplifies genes at constant temperatures

Question 21

How does RT-LAMP work?

Answer 21

Does not need high quality RNA, 4-6 primers, Bst DNA polymerase with strand displacement activity to minimize time, addition of reverse transcriptase

Question 22

What is the Nanomaterial based sensor?

Answer 22

Sensor composed of gold nanoparticles linked to organic ligands
Creates diverse sensing layer that shrinks when exposed to VOCs
Viral agents emit VOCs that could indicate early infection, leading to immediate infection