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Validation of a real-time polymerase chain reaction assay for sensitive quantification of francisella tularensis straiins

Beary, Megan (2018) Validation of a real-time polymerase chain reaction assay for sensitive quantification of francisella tularensis straiins. Master Essay, University of Pittsburgh.

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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.


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Details

Item Type: Other Thesis, Dissertation, or Long Paper (Master Essay)
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Beary, Meganmeb230@pitt.edumeb230
Contributors:
ContributionContributors NameEmailPitt UsernameORCID
Committee ChairReed, Dougdsreed@pitt.edudsreedUNSPECIFIED
Committee MemberHartman, Amyhartman2@pitt.eduUNSPECIFIEDUNSPECIFIED
Committee MemberMartinson, Jeremyjmartins@pitt.eduUNSPECIFIEDUNSPECIFIED
Centers: Other Centers, Institutes, or Units > Center for Vaccine Research
Date: 13 April 2018
Date Type: Submission
Number of Pages: 46
Institution: University of Pittsburgh
Schools and Programs: Graduate School of Public Health > Infectious Diseases and Microbiology
Degree: MPH - Master of Public Health
Thesis Type: Master Essay
Refereed: Yes
Date Deposited: 09 Nov 2018 14:49
Last Modified: 09 Nov 2018 14:49
URI: http://d-scholarship.pitt.edu/id/eprint/34082

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