1. Luciferase Based Plasmid Reporter System for the Detection and Quantification of Human Respiratory Syncytial Virus
Progress Report 1, 12/6/2007
Melanie Aston, Michael Chi, Monica Deterding, Matt Huckabee

2. Background
Human Respiratory Syncytial Virus is the most common cause of bronchiolitis and pneumonia in children under 1 year of age (CDC)
~ children die per year due to RSV infection, which is about 91 per hour
There is no current vaccine available for hRSV
Need quick cheap way to quantify RSV
Oral Report 1
Wednesday, November 14, 2018

3. Current Method: Viral Plaque Assay
Procedure
Culture cells in 12 well plates (2 per sample)
Infect cells
Continue culturing cells in media with methyl cellulose for 5 days
Stain with hematoxylin and eosin
RSV Sample
1:1
1:10
1:102
1:103
Talk about the “system” as a whole, meaning the plasmid and how to use it
Oral Report 1
Wednesday, November 14, 2018 3

4. Our Solution Novel plasmid based reporter system
A luciferase plasmid and cell line that will luminesce when infected with RSV
Stable transfection of plasmid into cell
Optimization of system protocol
Talk about the “system” as a whole, meaning the plasmid and how to use it
Oral Report 1
Wednesday, November 14, 2018

5. Methods Remove luciferase gene from pGEM-luc Oral Report 1
Wednesday, November 14, 2018

6. Methods Ligate luciferase and additional sequence together
Blue: leader, NS1 gene start, and non-coding regions
Red: non-coding, L gene end, and trailer regions
Oral Report 1
Wednesday, November 14, 2018

7. Methods
Cut pcDNA3.1. Ligate luciferase, additional sequence, and pcDNA3.1 together
Oral Report 1
Wednesday, November 14, 2018

8. Methods Transfect cells with plasmid Plasmid Oral Report 1
Wednesday, November 14, 2018

9. Methods Plate cells onto a 96-well plate Tests run in
triplicate for each concentration
Pipette cells into rows/columns of plate
Oral Report 1
Wednesday, November 14, 2018

10. Methods Infect cells with various RSV concentrations mRNA mRNA
Luciferase
Luciferin
Oral Report 1
Wednesday, November 14, 2018

11. Plate Reader Methods Measure luminescence Oral Report 1
Wednesday, November 14, 2018

12. Comparison: Evaluation Chart
Plaque Assay
Luciferase System
Criteria
Weight (1-5)
Value
Product
Quick 5 2 10 4 20
Low Cost 3
Objective 25
Efficient 15
Total 55 90
Oral Report 1
Wednesday, November 14, 2018

13. Comparison Plaque Assay Luciferase System Detection Method
Staining/Counting
Luminescence
Objectivity
Partial
Yes
Time (work / total)
10 hours / 7 days
2.5 hours / 2 days
Materials Cost $8 $1
Efficiency
30 samples/experiment
240 samples/experiment
Oral Report 1
Wednesday, November 14, 2018

14. Current Progress Completed: In Progress: Literature research
Laboratory training
Proof of Concept experiment
Purchased stock plasmids
In Progress:
Design of insertion sequences
Maxiprep purchased plasmids and create glycerol stocks
Oral Report 1
Wednesday, November 14, 2018

15. Future Work Finalize insertion sequences and test on computer
Order insertion sequences
Remove luciferase gene from pGEM-luc
Ligate insertion sequences and luciferase into pcDNA3.1
Test luminescence of cells using varying amounts of RSV
Optimizing the system
Oral Report 1
Wednesday, November 14, 2018