1. Luciferase Based Plasmid Reporter System for the Detection and Quantification of Human Respiratory Syncytial Virus
Group 14: Oral Report 3, 2/12/2008
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 (~91 per hour) due to RSV infection
There is no current vaccine available for RSV
Current method for quantification of infectious RSV: Plaque Assay
VUSE Senior Design
Oral Report 3
Tuesday, February 12, 2008

3. The Problem Viral plaque assay is Labor intensive Costly
Time consuming
Partially subjective
Need high throughput, inexpensive system to quantify infectious RSV
VUSE Senior Design
Oral Report 3
Tuesday, February 12, 2008

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 3
Tuesday, February 12, 2008

5. Comparison Plaque Assay Luciferase System Detection Method
Staining/Counting
Luminescence
Objectivity
Partial
Yes
Time (work/total)
10 hours/7 days
2.5hrs/2 days
Materials Cost $8 $1
Throughput
30 samples/experiment
240 samples/experiment
VUSE Senior Design
Oral Report 3
Tuesday, February 12, 2008

6. 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
VUSE Senior Design
Oral Report 3
Tuesday, February 12, 2008

7. Methods RSV Genome RSV Genome (truncated) NS1 Start (Synthesized)SH M2 3’ 5’ N M G L F P
RSV Genome (truncated)
NS1 L 3’ 5’
NS1 Start
L Stop
pcDNA
(Synthesized)

8. Methods
Luciferase Gene (luc)
L Stop
NS1 Start
luc
pRSVlucM5
selection

9. pRSVlucM5
VUSE Senior Design
Oral Report 3
Tuesday, February 12, 2008

10. Development Costs Item Cost pcDNA3.1 vector $361 pGEM-luc $83
Trailer minigenome plasmid
$274
Leader oligonucleotides
2x at $78 and 2x at $98
Cloning discs
2x at $29
Misc. chemicals and disposable lab equip.
$750*
TOTAL
$1878*
* Indicates an approximate value, many supplies are for general lab use
VUSE Senior Design
Oral Report 3
Tuesday, February 12, 2008

11. Factors Affecting Success
There are 5 possible plasmids resulting from the combination of our four DNA molecules; we must screen for the correct one: pRSVlucM5
Unforeseen problems with designed sequences
Sensitivity relative to plaque assay
VUSE Senior Design
Oral Report 3
Tuesday, February 12, 2008

12. Alternate Solutions PCR - polymerase chain reaction Limitations:
Proven to work for the detection and quantification of viruses
Limitations:
Measures amount of nucleic acid (cannot differentiate between live virus and dead virus)
Low throughput
Costly
VUSE Senior Design
Oral Report 3
Tuesday, February 12, 2008

13. Current Progress Completed: In Progress:
Design of all plasmid constituents in silco
Purified all plasmid constituents by gel electrophoresis
Quantify all four sequences
Ligate three sequences into pcDNA3.1vector
Transform e. coli with plasmids
Screen colonies with minipreps
In Progress:
Maxiprep correct colony to obtain high yield of final plasmid
Submit Information Disclosure forms to Office of Tech Transfer
VUSE Senior Design
Oral Report 3
Tuesday, February 12, 2008

14. Screening Cut with SphI VUSE Senior Design Oral Report 3
1146bp
795bp
574bp
4908bp
72bp
VUSE Senior Design
Oral Report 3
Tuesday, February 12, 2008

15. Future Work Stably transfect cells with final plasmid
Test luminescence of cells using varying amounts of RSV
Optimize the system
VUSE Senior Design
Oral Report 3
Tuesday, February 12, 2008