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Luciferase Based Plasmid Reporter System for the Detection and Quantification of Human Respiratory Syncytial Virus Group 14: Oral Report 3, 2/12/2008 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 (~91 per hour) due to RSV infection There is no current vaccine available for RSV Current method for quantification of infectious RSV: Plaque Assay VUSE Senior Design Oral Report 3 Tuesday, February 12, 2008 The Problem 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 3 Tuesday, February 12, 2008 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 3 Tuesday, February 12, 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 3 Tuesday, February 12, 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 3 90 Tuesday, February 12, 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 3 Tuesday, February 12, 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* * Indicates an approximate value, many supplies are for general lab use VUSE Senior Design Oral Report 3 Tuesday, February 12, 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 Unforeseen problems with designed sequences Sensitivity relative to plaque assay VUSE Senior Design Oral Report 3 Tuesday, February 12, 2008 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 VUSE Senior Design Oral Report 3 Tuesday, February 12, 2008 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 VUSE Senior Design Oral Report 3 Tuesday, February 12, 2008 Screening Cut with SphI 1146bp 795bp 574bp 4908bp 72bp VUSE Senior Design Oral Report 3 Tuesday, February 12, 2008 Future Work Stably transfect cells with final plasmid Test luminescence of cells using varying amounts of RSV Optimize the system VUSE Senior Design Oral Report 3 Tuesday, February 12, 2008