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