Download - Group 14: Oral Report 2, 1/24/2008 Melanie Aston, Michael Chi, Monica Deterding, Matt Huckabee
Luciferase Based Plasmid Reporter System for the Detection and Quantification of
Human Respiratory Syncytial Virus
Group 14: Oral Report 2, 1/24/2008Melanie 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 due to RSV infection, which is about 91 per hour
There is no current vaccine available for RSV Current method for quantification of infectious RSV:
Plaque Assay
Background
Thursday, January 24, 2008VUSE Senior Design Oral Report 2
The Problem Viral plaque assay is
Labor intensive Costly Time consuming Partially subjective
Need high throughput, inexpensive system to quantify infectious RSV
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
Thursday, January 24, 2008VUSE Senior Design Oral Report 2
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
Thursday, January 24, 2008VUSE Senior Design Oral Report 2
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
Efficiency 30 samples/experiment 240 samples/experiment
Thursday, January 24, 2008Oral Report 2VUSE Senior Design
Methods Remove luciferase gene from pGEM-luc
Thursday, January 24, 2008VUSE Senior Design Oral Report 2
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
Thursday, January 24, 2008VUSE Senior Design Oral Report 2
Cut pcDNA3.1. Ligate luciferase, additional sequences, and pcDNA3.1 together
Methods
Thursday, January 24, 2008VUSE Senior Design Oral Report 2
pRSVlucM5
Thursday, January 24, 2008Oral Report 2VUSE Senior Design
Transfect cells with plasmid
Methods
Plasmid
Thursday, January 24, 2008VUSE Senior Design Oral Report 2
Infect cells with various RSV concentrations
Methods
mRNA
Luciferase
mRNA
Luciferin
Thursday, January 24, 2008VUSE Senior Design Oral Report 2
Measure luminescence
Methods
Plate Reader
Thursday, January 24, 2008VUSE Senior Design Oral Report 2
Development CostsItem Cost
pcDNA3.1 vector $361.00
pGEM-luc $83.00
Trailer minigenome plasmid $274
Leader oligonucleotides 2x at $78.00 and 2x at $97.50
Cloning discs 2x at $29.30
Misc. chemicals and disposable lab equip. $750*
TOTAL $1877.60*
Thursday, January 24, 2008Oral Report 2VUSE Senior Design
* Indicates an approximate value, many supplies are for general lab use
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
Possible E. coli rejection of RSV sequences Sensitivity relative to plaque assay
Thursday, January 24, 2008Oral Report 2VUSE Senior Design
Alternate Solutions Try other E. coli strains 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
Thursday, January 24, 2008VUSE Senior Design Oral Report 2
Current Progress Completed:
Design of leader and trailer sequences Design of final plasmid construct in silco Purified pcDNA3.1 vector and luciferase insert
In Progress: Preparation of leader and trailer inserts Gel purification of leader and trailer inserts
Thursday, January 24, 2008VUSE Senior Design Oral Report 2
Setbacks 1/18/08
Failure of oligonucleotide ligation due to unknown factors
Failure of trailer double digest due to unknown factors
1/21/08 Confirmation of ligation
failure due to lack of 5’ phosphorylation
Success of trailer double digest
1/18
1/21
Thursday, January 24, 2008VUSE Senior Design Oral Report 2
Future Work Phosphorylate and ligate leader insert parts Cut out trailer insert from minigene plasmid Quantify all four sequences Ligate three sequences into pcDNA3.1vector Transform e. coli with plasmids Screen colonies with minipreps Maxiprep correct colony to obtain high yield of final plasmid Stably transfect cells with final plasmid Test luminescence of cells using varying amounts of RSV Optimize the system
Thursday, January 24, 2008VUSE Senior Design Oral Report 2