Transcript Luciferase Based Plasmid Reporter System for the

Luciferase Based Plasmid Reporter System for the Detection and
Quantification of
Human Respiratory Syncytial Virus
Group 14: Oral Report 4, 3/13/08
Melanie Aston, Michael Chi, Monica Deterding, Matt Huckabee
Background on RSV in the Clinic
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Human Respiratory Syncytial Virus is the most
common cause of bronchiolitis and pneumonia in
children under 1 year of age (CDC)
About 800000 children die per year worldwide due to
RSV infection (~91 per hour)
There are currently two methods for the clinical
confirmation of RSV infection
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Viral isolation from culture (gold standard, requires several days)
Direct antigen test (tests range from 20-75 mins)
There are currently no vaccines or drugs available to
prevent or treat RSV
VUSE Senior Design
Oral Report 4
Thursday March 13th, 2008
Background on RSV in the Lab
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Ongoing RSV research:
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Understand mechanisms of RSV pathogenesis in order to
develop drugs
Test vaccine candidates
Mouse models are commonly used
The current method to quantify RSV titer in mice is
the plaque assay
VUSE Senior Design
Oral Report 4
Thursday March 13th, 2008
Current Method: Viral Plaque Assay
Culture
Cells
Wait
For Cells to
Grow
Inoculate
Cells with
Virus
Wait for Cells
to become
Infected
3 days
Overlay Cells
with MethylCellulose
1 hour
Allow
Plaques
To Form
Stain Cells with
Hematoxylin
and Eosin
5 days
Count
Plaques
VUSE Senior Design
Calculate
Viral
Titer
Oral Report 4
Thursday March 13th, 2008
The Problem
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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 4
Thursday March 13th, 2008
Our Solution
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Novel plasmid based reporter system
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Luciferase plasmid
Cell line
Luminesce upon infection with RSV
VUSE Senior Design
Oral Report 4
Thursday March 13th, 2008
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 4
Thursday March 13th, 2008
Comparison: Evaluation Chart
Plaque Assay
Criteria
Weight (1-5)
Value
Luciferase System
Product
Value
Product
Quick
5
2
10
4
20
Low Cost
3
2
6
4
12
Objective
3
4
12
5
15
Efficient
4
3
12
5
20
Total
VUSE Senior Design
40
Oral Report 4
67
Thursday March 13th, 2008
Methods
RSV Genome
NS1NS2
N
3’
SH
P
M
M2
G
F
L
5’
RSV Genome (truncated)
NS1
3’
L
NS1 Start
5’
L Stop
pcDNA
(Synthesized)
Methods
Luciferase Gene (luc)
L Stop
NS1 Start
luc
pRSVlucM5
selection
pRSVlucM5
VUSE Senior Design
Oral Report 4
Thursday March 13th, 2008
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*
* Approximate value
VUSE Senior Design
Oral Report 4
Thursday March 13th, 2008
Alternate Solutions
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PCR - polymerase chain reaction
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Proven to work for the detection and quantification of
viruses
Limitations:
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Measures amount of nucleic acid (cannot differentiate
between live virus and dead virus)
Low throughput
Costly
VUSE Senior Design
Oral Report 4
Thursday March 13th, 2008
Project Status
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Completed:
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Design of all plasmid constituents in silco
Ligation of the plasmid constituents
Screening selection
Demonstration that the plasmid works as designed
Stable transfection of cells with plasmid
Submitted Information Disclosure Form to Office of
Tech Transfer
VUSE Senior Design
Oral Report 4
Thursday March 13th, 2008
Luminescence Data
RLU vs. Amount of DNA Transfected
3000
2500
RLU
2000
(-) control
2,1
1500
2,2
3,1
1000
3,2
500
0
0
0.2
0.4
0.6
0.8
1
Amt of DNA Transfected (ug)
VUSE Senior Design
Oral Report 4
Thursday March 13th, 2008
Project Status
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In Progress:
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Test stable transfection
Future Work:
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Optimize the system
Final write-up
VUSE Senior Design
Oral Report 4
Thursday March 13th, 2008