Luciferase Based Plasmid Reporter System for the Detection and Quantification of
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Group 14: Oral Report 4, 3/13/08 Melanie Aston, Michael Chi, Monica Deterding, Matt Huckabee - PowerPoint PPT Presentation


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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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Luciferase Based Plasmid Reporter System for the Detection and Quantification ofHuman 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
Background on RSV in the Clinic and Quantification of

  • 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

    • 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
Background on RSV in the Lab and Quantification of

  • Ongoing RSV research:

    • 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
Current Method: Viral Plaque Assay and Quantification of

Culture

Cells

Wait

For Cells to

Grow

Inoculate

Cells with

Virus

Wait for Cells

to become

Infected

3 days

1 hour

Overlay Cells

with Methyl-

Cellulose

Allow

Plaques

To Form

Stain Cells with

Hematoxylin

and Eosin

5 days

Count

Plaques

Calculate

Viral

Titer

VUSE Senior Design

Oral Report 4

Thursday March 13th, 2008


The problem
The Problem and Quantification of

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
Our Solution and Quantification of

  • Novel plasmid based reporter system

    • Luciferase plasmid

    • Cell line

  • Luminesce upon infection with RSV

VUSE Senior Design

Oral Report 4

Thursday March 13th, 2008


Comparison
Comparison and Quantification of

VUSE Senior Design

Oral Report 4

Thursday March 13th, 2008


Comparison evaluation chart
Comparison: Evaluation Chart and Quantification of

VUSE Senior Design

Oral Report 4

Thursday March 13th, 2008


Methods
Methods and Quantification of

NS1

NS2

SH

M2

P

N

M

G

F

L

3’

5’

NS1

L

3’

5’

NS1 Start

L Stop

pcDNA

RSV Genome

RSV Genome (truncated)

(Synthesized)


Methods1
Methods and Quantification of

Luciferase Gene (luc)

L Stop

NS1 Start

luc

pRSVlucM5

selection


Prsvlucm5
pRSVlucM5 and Quantification of

VUSE Senior Design

Oral Report 4

Thursday March 13th, 2008


Development costs
Development Costs and Quantification of

* Approximate value

VUSE Senior Design

Oral Report 4

Thursday March 13th, 2008


Alternate solutions
Alternate Solutions and Quantification of

  • 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

VUSE Senior Design

Oral Report 4

Thursday March 13th, 2008


Project status
Project Status and Quantification of

  • Completed:

    • 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
Luminescence Data and Quantification of

VUSE Senior Design

Oral Report 4

Thursday March 13th, 2008


Project status1
Project Status and Quantification of

  • In Progress:

    • Test stable transfection

  • Future Work:

    • Optimize the system

    • Final write-up

VUSE Senior Design

Oral Report 4

Thursday March 13th, 2008


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