eprintid: 34082
rev_number: 19
userid: 7238
dir: disk0/00/03/40/82
datestamp: 2018-11-09 14:49:02
lastmod: 2018-11-09 14:49:02
status_changed: 2018-11-09 14:49:02
type: thesis
metadata_visibility: show
contact_email: megan.beary@gmail.com
eprint_status: archive
creators_name: Beary, Megan
creators_email: meb230@pitt.edu
creators_id: meb230
contributors_type: committee_chair
contributors_type: committee_member
contributors_type: committee_member
contributors_name: Reed, Doug
contributors_name: Hartman, Amy
contributors_name: Martinson, Jeremy
contributors_email: dsreed@pitt.edu
contributors_email: hartman2@pitt.edu
contributors_email: jmartins@pitt.edu
contributors_id: dsreed
title: Validation of a real-time polymerase chain reaction assay for sensitive quantification of francisella tularensis straiins
ispublished: unpub
divisions: sch_gsph_infectiousdiseasesmicrobiology
full_text_status: public
abstract: Tularemia, caused by the Gram-negative bacterium Francisella tularensis, is a life-threatening human and animal disease. F. tularensis has a wide geographical distribution, a variety of vectors and reservoirs, and a low infectious dose, causing it to be considered a potential biological weapon and public health threat. There is currently no FDA-approved vaccine for F. tularensis. A sensitive and specific quantification method is needed to accurately detect and quantify bacterial isolates. A review of the literature has shown that real-time PCR can more sensitively quantify F. tularensis genomes than traditional plating methods. In order to contribute to the development of a vaccine, we have done the following: evaluated four sets of primers for use in real-time PCR with F. tularensis, enhanced the genomic DNA extraction protocol with an extra spin step and with 10 minutes added to the incubation period to make it more efficient, and enhanced quantification done by the Nanodrop™ One Spectrophotometer by heating samples to 63⁰C before reading.  The evaluation of primers and the method validation of genomic DNA extraction and quantification will allow us to develop PCR standards for four subspecies of F. tularensis initially, and eventually will allow us to quantify bacterium in infected animal tissues.  The public health significance of this work is to further vaccine development for an extremely infectious disease in humans and animals.
date: 2018-04-13
date_type: submitted
pages: 46
institution: University of Pittsburgh
refereed: TRUE
centers: cen_other_vaccineresearch
thesis_type: masteressay
degree: MPH
citation:   Beary, Megan  (2018) Validation of a real-time polymerase chain reaction assay for sensitive quantification of francisella tularensis straiins.  Master Essay, University of Pittsburgh.   
document_url: http://d-scholarship-dev.library.pitt.edu/34082/1/BearyMegan_MPHessay_April2018.doc