Luciferase Based Plasmid Reporter System for the Detection and Quantification of
Human Respiratory Syncytial Virus
Group 14: Oral Report 3, 2/12/2008Melanie Aston, Michael Chi, Monica Deterding, Matt Huckabee
Human Respiratory Syncytial Virus is the most common cause of bronchiolitis and pneumonia in children under 1 year of age (CDC)
~800000 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
Background
Tuesday, February 12, 2008VUSE Senior Design Oral Report 3
The Problem Viral plaque assay is
Labor intensive Costly Time consuming Partially subjective
Need high throughput, inexpensive system to quantify infectious RSV
Tuesday, February 12, 2008VUSE Senior Design Oral Report 3
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
Tuesday, February 12, 2008VUSE Senior Design Oral Report 3
ComparisonPlaque 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
Tuesday, February 12, 2008VUSE Senior Design Oral Report 3
Comparison: Evaluation Chart
Plaque Assay Luciferase System
Criteria Weight (1-5) Value Product Value Product
Quick 5 2 10 4 20
Low Cost 3 2 10 4 20
Objective 3 4 20 5 25
Efficient 4 3 15 5 25
Total 55 90
Tuesday, February 12, 2008VUSE Senior Design Oral Report 3
MethodsRSV Genome
RSV Genome (truncated)NS1
L3’ 5’
NS1 Start L Stop
pcDNA
(Synthesized)
NS1NS2 SH M2
3’ 5’N M G LFP
Methods
Luciferase Gene (luc)
luc
NS1 Start L Stop
selectionpRSVlucM5
pRSVlucM5
Tuesday, February 12, 2008VUSE Senior Design Oral Report 3
Development CostsItem 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
Tuesday, February 12, 2008VUSE Senior Design Oral Report 3
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
Tuesday, February 12, 2008VUSE Senior Design Oral Report 3
Alternate Solutions PCR - polymerase chain reaction
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
Tuesday, February 12, 2008VUSE Senior Design Oral Report 3
Current Progress Completed:
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
Tuesday, February 12, 2008VUSE Senior Design Oral Report 3
Screening
Tuesday, February 12, 2008VUSE Senior Design Oral Report 3
72bp
4908bp
1146bp
795bp
574bp
Cut with SphI
Future Work Stably transfect cells with final plasmid Test luminescence of cells using varying amounts of RSV Optimize the system
Tuesday, February 12, 2008VUSE Senior Design Oral Report 3