Document Type

Thesis

Degree

Master of Science (MS)

Department

Forensic Science

First Advisor's Name

Kalai Mathee

First Advisor's Committee Title

Committee Chair

Second Advisor's Name

DeEtta Mills

Third Advisor's Name

Giri Narasimhan

Keywords

Biology, Forensics, DNA, Real-time qPCR, Biological agents, Detection

Date of Defense

11-15-2013

Abstract

Real-time quantitative polymerase chain reaction (real-time qPCR) assays are an effective technique to detect biological warfare agents and surrogate organisms. In my study, primers were designed to detect chromosomal DNA of biological warfare agent surrogates B. thuringiensis and S. marcescens (representing B. anthracis and Y. pestis, respectively) via real-time qPCR. Species-level specificity of the primers was demonstrated through comparisons with a bacterial strain panel and corroborated by qPCR data. Additionally, the primer efficacy was tested when template DNA was spiked into metagenomic DNA extracted from clinical lung microbiome samples. The results showed that while detection of B. thuringiensis or S. marcescens was still largely successful, the addition of metagenomic DNA did significantly inhibit amplification in most cases. The present study is significant not only for the design of multiple novel primer pairs able to detect bacterial agents in metagenomic DNA, but also the quantitative insight to the influence of background DNA on single species detection at low DNA concentrations.

Identifier

FI13121204

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