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Luciferase Based Plasmid Reporter System for the Detection and Quantification of Human Respiratory Syncytial Virus Group 14: Oral Report 2, 1/24/2008 Melanie Aston, Michael Chi, Monica Deterding, Matt Huckabee Background 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 VUSE Senior Design Oral Report 2 Thursday, January 24, 2008 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 VUSE Senior Design Oral Report 2 Thursday, January 24, 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 10 4 20 Objective 3 4 20 5 25 Efficient 4 3 15 5 25 Total VUSE Senior Design 55 Oral Report 2 90 Thursday, January 24, 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 Efficiency 30 samples/experiment 240 samples/experiment VUSE Senior Design Oral Report 2 Thursday, January 24, 2008 Methods Remove luciferase gene from pGEM-luc VUSE Senior Design Oral Report 2 Thursday, January 24, 2008 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 VUSE Senior Design Oral Report 2 Thursday, January 24, 2008 Methods Cut pcDNA3.1. Ligate luciferase, additional sequences, and pcDNA3.1 together VUSE Senior Design Oral Report 2 Thursday, January 24, 2008 pRSVlucM5 VUSE Senior Design Oral Report 2 Thursday, January 24, 2008 Methods Transfect cells with plasmid Plasmid VUSE Senior Design Oral Report 2 Thursday, January 24, 2008 Methods Infect cells with various RSV concentrations mRNA mRNA Luciferase Luciferin VUSE Senior Design Oral Report 2 Thursday, January 24, 2008 Methods Measure luminescence Plate Reader VUSE Senior Design Oral Report 2 Thursday, January 24, 2008 Development Costs Item 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* * Indicates an approximate value, many supplies are for general lab use VUSE Senior Design Oral Report 2 Thursday, January 24, 2008 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 VUSE Senior Design Oral Report 2 Thursday, January 24, 2008 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 VUSE Senior Design Oral Report 2 Thursday, January 24, 2008 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 VUSE Senior Design Oral Report 2 Thursday, January 24, 2008 Setbacks 1/18/08 Failure of oligonucleotide ligation due to unknown factors Failure of trailer double digest due to unknown factors 1/18 1/21/08 Confirmation of ligation failure due to lack of 5’ phosphorylation Success of trailer double digest 1/21 VUSE Senior Design Oral Report 2 Thursday, January 24, 2008 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 VUSE Senior Design Oral Report 2 Thursday, January 24, 2008