Development of isolation and detection methods for the analysis of DNA and bacteria

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Varona, Marcelino
Major Professor
Anderson, Jared L
Nilsen-Hamilton, Marit
Lee, Young-Jin
Venditti, Vincenzo
Anand, Robbyn
Committee Member
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Nucleic acids are essential biopolymers that have served as diagnostic biomarkers for many applications. Traditional nucleic acid isolation methods typically rely on nucleic acid adsorption onto silica particles and require multiple centrifugation steps, organic solvents and significant user intervention. Additionally, popular detection methods, such as polymerase chain reaction (PCR), suffer from long analysis times and require complex thermal cycling equipment. These drawbacks limit the utility of traditional isolation/detection methods for rapid in-field testing and point-of-care applications. The work presented in this dissertation addresses challenges associated with nucleic acid isolation and detection through the development of solid-phase microextraction (SPME) and isothermal amplification methodologies. SPME is an alternative sample preparation methodology that circumvents many limitations of conventional methods. A rapid SPME method was developed and optimized to enable the isolation of DNA from mycobacterium smegmatis in artificial sputum samples. For detecting the extracted DNA, a colorimetric isothermal multiple-self-matching initiated amplification (IMSA) assay was designed using hydroxy napthol blue (HNB). A custom buffer was designed to allow direct coupling of the 1 M NaCl SPME desorption solution with the IMSA assay. The custom buffer was successfully applied in multiple studies for the detection of a variety of DNA sequences. A significant limitation of isothermal amplification methodologies is the lack of sequence-specificity in the detection method. Traditional methods rely on colorimetric indicators that indirectly detect amplification. Molecular beacons (MB) were employed along with HNB to specifically detect loop-mediated isothermal amplification (LAMP) using a transilluminator. The high specificity of the method was demonstrated by discriminating two sequences that varied by a single nucleotide. To further demonstrate the applicability of MB-LAMP, an assay was designed for the detection of BRAF V600E, a mutation present in 90% of melanomas. Two MBs were employed containing a FAM (wild-type) or HEX (mutant) fluorophore to identify each sequence. A plate reader was employed in order to simply the detection scheme. Additionally, SPME was successfully applied to isolate the mutant sequence from human plasma and enable detection with the MB-LAMP assay. The specific detection of LAMP was further simplified by employing MBs and a biotinylated primer in a lateral-flow immunoassay format. This approach eliminated the need for additional equipment in the detection step. The versatility of the method was demonstrated through the detection of three independent sequences. Additionally, the high specificity of the method was highlighted through the differentiation of wild type and mutant BRAF V600E. The developed detection scheme was also compatible with SPME and enabled detection from pond water, human plasma, and artificial saliva. A disadvantage to the previously used SPME extraction phases is the lack of specificity, as the primary interaction with the nucleic acid was anion exchange. To circumvent this, a sequence-specific SPME sorbent was developed in collaboration with Millipore Sigma. A commercially-available polyacrylate SPME fiber was modified with an amine-labeled oligonucleotide complementary to a DNA target using carbodiimide coupling chemistry. Several parameters were optimized including desorption time and the use of Exonuclease III to minimize carryover. While detection of nucleic acids from pathogens is often sufficient in many cases, certain applications such as food safety require knowledge of the viability of the detected organisms. Traditional methods rely on pre-enrichment cultures that are time consuming to perform, decrease the sample-to-answer time. Magnetic ionic-liquids have been previously shown to be capable of enriching viable bacteria from a variety of food matrices. In the study presented within this dissertation, the physiological effects of magnetic ionic liquids on a variety of bacteria were evaluated. Salmonella and E. coli O157:H7 that were captured by magnetic ionic liquids were plated on selective and non-selective agars to evaluate whether or not cellular injury occurred. Additionally, the ability for magnetic ionic liquids to capture a wide range of Gram-negative bacteria was also evaluated.
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