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Escherichia coli O157:H7 detection in food is conducted mainly by DNA/PCR, immunoassay or conventional methods. However, all the methods require multiple incubation steps. Antibiotic and isolation agars were found as the main factors that lead to false-positive results. An improved rapid detection method was developed by decreasing detection time and enhancing easiness of detection without the need for any analytical instrumentation. A combination of selective ingredients and temperature was utilized to allow the growth of Escherichia coli O157:H7 in the detection. The detection method minimized the effects of the main false positive bacteria, Pseudomonas spp. and Enterobacter spp. The sensitivity, specificity and accuracy of the 24h detection method in foodstuffs were 96.2%, 99.6% and 97.0%, respectively when the original inoculation was 10-100cfu/g in food. This method can be utilized to detect Escherichia coli O157:H7 in foodstuffs more rapidly, economically and conveniently when compared to the methods that are currently used.

Listeria monocytogenes is a foodbome pathogen that can cause severe illness and even death. It is found in dairy and meat products. The focus is on rapid detection since conventional methods are time consuming (4-5 days). Pre-enrichment steps, as part of those methods, are time consuming. Our objective was to develop a detection system without a pre-enrichment step, giving a final result within 2 to 4 h. In the concept of "the need for speed," a detection system with an antibody-based capture technique, followed by polymerase chain reaction (PCR), was developed. Glass beads coated with a Listeria polyclonal antibody were added to the food sample. After a static incubation/capturing step, beads-cell complexes were separated from the food, and boiled to lyse the cells and release the DNA. In a final PCR/electrophoresis step the DNA samples were analyzed. The use of a flow-based capturing system (ImmunoFlow) was also investigated. Using a bead-antibody complex in this ImmunoFlow setup has several advantages, including the possibility of concentrating the microorganisms out of large food samples (with flow through setup), the exclusion of a pre-enrichment step, and the potential for automation. Besides buffer solution (Tris), different kinds of milk, e.g., pasteurized, Ultra High Temperature (UHT), and raw milk, were also investigated. The detection limit in buffer solution was 1 x 106 CFU/ml no matter if the ImmunoFlow system or the static incubation was used. For the different pasteurized milk samples, the detection limit varied between 1 x 107 and 1 x 108 cells/ml in the static procedure. For UHT and raw milk, however, capturing of Listeria monocytogenes cells was not possible in the static or the ImmunoFlow setup. In conclusion, we developed a rapid and specific detection system for Listeria monocytogenes at high concentration in pasteurized milk using a static capturing procedure. The total test time for this detection system is less than 4 h, which is much faster than the present detection systems (which are using an enrichment step prior to testing). Implementing a real-time PCR system after capture would further reduce this detection time.

Qualitative and quantitative analysis of trace substances determined by Mass spectrometry has unique advantages which can¡¦t be replaced. For example, the detection limit of common gas sensors are difficult to lower than 1 ppm, and the sensitivity, selectivity, period of use and stability are not ideal. The detection limit of mass spectrometer is general low to 0.01 ppm. Furthermore, all substances in the sample can be simultaneous analysis by mass spectrometer, but single gas sensor measurements cannot. In this study, dielectric barrier discharge plasma is used in environmental mass spectrometry analysis. This study develops an innovative balanced T-shaped dielectric barrier discharge (DBD) plasma generator for generating atmospheric plasma to replace the linear type plasma generator. Through the change of the geometric configuration and the drive phase develop T-shaped dielectric barrier discharge plasma, the balanced design can fully cancel the high potential and noise. The main objective of this study for the more traditional linear electrodes of the study's original novel T-shaped electrodes of different, including mass spectrometry, spectroscopy, and some basic electrical measurements, and by changing the electrode design, voltage, temperature, gas flow, gas flow rate, diameter and other parameters of the dielectric, and compare their differences and to explore the most suitable parameters. The results showed that T-shaped design of the research and development of innovation through the elimination of the exit pressure put EFI flame can indeed significantly reduce sample oxidation and generate fragments of the situation, and thus improve the mass spectrum of readability and debris interference, thus improving the detection limit , especially for some with a benzene ring and long-chain carbon samples. The experimental results confirm that the development of the Institute of Atmospheric Pressure Plasma free system can be prolonged to produce high concentrations of plasma gas as a free source of the mass spectrometry system, and provides more than 107 cm-3 ion concentration. MS-free analysis of the system can be directly on the gas, liquid and solid samples, the test do not need complicated traditional mass spectrometry analysis of the required sample pre-treatment steps, you can get a clear identification of high mass spectrometry signal. In addition to introducing the basic principles and structure of the atmospheric pressure plasma discharge device, and take advantage of many samples test for the different plasma mass spectrometry free system performance verification.

To protect public health, detection and treatment technologies have been improved to monitor and inactivate pathogens in drinking water. The goal of this dissertation is to evaluate and utilize multiple online sensors and advanced oxidation processes to document both the detection as well as destruction of microbial contaminants in real-time. Reviews of rapid detection technologies for real-time monitoring of pathogens in drinking water and advanced technologies to inactivate pathogens in water are shown in Appendices A and B. The study in Appendix C evaluated the efficacy of real-time sensors for the detection of microbial contaminants. Bacillus thuringiensis was used in this research as a surrogate for Bacillus anthracis to determine each sensor response and detection capability. The minimum threshold responses of sensors were determined by injecting B.thuringiensis into deionized (DI), raw (unfiltered) tap water, or filtered tap water over a concentration range of 10² - 10⁵ spores/ml. The BioSentry sensor responded to increases in concentration over the range of 10² - 10⁵ spores/ml. Below this range, sensors provided signals undistinguishable from background noise. The select sensors can detect microbial water quality changes, and these advanced technologies can be integrated to monitor intrusion events in water distribution systems. The study in Appendix D evaluated the efficiency of the UV reactor for inactivation of MS2 coliphage. The virus MS2 coliphage (ATCC 15597-B1) has been proposed by the U.S. Environmental Protection Agency as a standard for UV reactor validation in the United States. In addition, MS2 is used as a surrogate for enteric viruses due to its similar size and morphology. Following UV radiation at a flow rate of 2gpm, infective MS2 showed a reduction of 5.3- log₁₀ when quantified with cultural plaque counts, whereas corresponding quantitative polymerase chain reaction (qPCR) data showed only a 1.7- log₁₀ reduction in viral RNA copy number. In contrast, plaque assay revealed a 5.8- log₁₀ inactivation; a slight increase in infective MS2 coliphage reduction at 1 gal per min but qPCR results indicate a 2.8- log₁₀ reduction in viral RNA copy number; a one log more inactivation compared to 2 gpm. When H₂O₂ was added at either 2.5 or 5 mg/l with UV at either flow rate, enhanced MS2 inactivation occurred with a greater than 7 log₁₀ reduction observed via plaque counts, indicating that all added MS2 had been inactivated, since no plaques were formed after incubation at 37°C for 24 hours. Correspondingly, qPCR data only showed a 3-4 log₁₀ reduction in viral RNA copy number. The study in Appendix E utilized online sensor to document the destruction of E.coli and Bacillus thuringiensis spores by UV/H₂O₂ treatment. In this study, Escherichia coli was tested for potential UV/H2O2 treatment in DI water and online sensors were also integrated to monitor the destruction in real-time. Pilot-scale experiments were performed using a Trojan UVSwift SC reactor (Trojan Technologies, London, ON, Canada) at a flow rate of 1 gal./min (gpm). UV radiation and UV/H₂O₂ combination in E.coli cell suspensions resulted in a >6 log₁₀ reduction of the viable counts. Similar exposure to B.thuringiensis spores resulted in a 3 log₁₀ reduction in viable counts. Scanning electron microscopy of the treated samples revealed severe damage on the surface of most E.coli cells, yet there was no significant change observed in the morphology of the B. thuringiensis spores. Following UV/H₂O₂ exposure, the BioSentry sensor showed an increase in the unknown, rod and spores counts, and did not correspond well when compared to viable counts assays. Data from this study show that advanced oxidation processes effectively inactivate E. coli vegetative cells, but not B.thuringiensis spores which were more resistant to UV/H₂O₂.

Current filter validation methods require 48 hours culture for results to become available, which creates time delays within the manufacturing process and quality control back-logs The thesis compares alternative methods for the production of filter challenge test data Within 24 hours to the desired test sensitivity, using bioluminescent and fluorescent genetically engineered strains of the test organism Brevundzmonas dzmznuta The recombinant strains were produced using a Tn5 transposon system, using a filter mating method. The genes cloned into the bacterial chromosome were the biolummescence lux_ genes, taken from the marme bacteria, Photorhabdus lummescens or Vzbno harveyz, and the gene encoding green fluorescent protein taken from the marine jelly fish Aequoria victoria The cloned strains were found to show no difference to the w1ld type strain With respect to their surface hydrophobicity, according to a bacterial adherence to hydrocarbons assay, and surface charge, according to an electro-static interaction chromatography method. Furthermore, the cell size according to Transmission Electron Microscopy was not significantly different to the wild type strain, which had cell dimensions of 1 05 x 0 52 Jlm The retention of cells by 0 45 mtcron rated filters, was shown to be not significantly different to the wild type All strains were retained by 0 2 Jlm filters These data confirmed that the cloned strains were suitable for challenge testing Four methods were used to detect microcolonies of the recombinant strains on filters. The advantage of the microcolony detection system was that it showed that the cells detected downstream of the filter were viable and culturable. The best detection method was with an epifluorescent microscope and the fluorescent strain after 24 hours, for which the sensitivity was 98.1 %. Two CCD camera systems were used to detect the bioluminescent strains on filters. The sensitivity of these systems were 80.1% and 83 9%, for the Nucleovision and Nightowl CCD camera systems, respectively, after 24 hours In addition, the Bwprobe photomultipherbased system was shown to achieve the detection sensitivity of one microcolony after 24 hours. Also, steps were made to study transcription Initiation signals for gene expression in fluorescent recombinant Brevundzmonas dzmmuta. Various putative promoter sequences were Identified in one fluorescent strain, using a DNA sequencmg method. These sequences showed homology to previously identified E colr and Brevundzmonas promoter sequences. Finally, an attempt was made to produce recombinant fluorescent and bioluminescent Acholeplasma lazdlawu, however this was unsuccessful and further work will be required to achieve this objective.

abstract: The need for rapid, specific and sensitive assays that provide a detection of bacterial indicators are important for monitoring water quality. Rapid detection using biosensor is a novel approach for microbiological testing applications. Besides, validation of rapid methods is an obstacle in adoption of such new bio-sensing technologies. In this study, the strategy developed is based on using the compound 4-methylumbelliferyl glucuronide (MUG), which is hydrolyzed rapidly by the action of E. coli β-D-glucuronidase (GUD) enzyme to yield a fluorogenic product that can be quantified and directly related to the number of E. coli cells present in water samples. The detection time required for the biosensor response ranged from 30 to 120 minutes, depending on the number of bacteria. The specificity of the MUG based biosensor platform assay for the detection of E. coli was examined by pure cultures of non-target bacterial genera and also non-target substrates. GUD activity was found to be specific for E. coli and no such enzymatic activity was detected in other species. Moreover, the sensitivity of rapid enzymatic assays was investigated and repeatedly determined to be less than 10 E. coli cells per reaction vial concentrated from 100 mL of water samples. The applicability of the method was tested by performing fluorescence assays under pure and mixed bacterial flora in environmental samples. In addition, the procedural QA/QC for routine monitoring of drinking water samples have been validated by comparing the performance of the biosensor platform for the detection of E. coli and culture-based standard techniques such as Membrane Filtration (MF). The results of this study indicated that the fluorescence signals generated in samples using specific substrate molecules can be utilized to develop a bio-sensing platform for the detection of E. coli in drinking water. The procedural QA/QC of the biosensor will provide both industry and regulatory authorities a useful tool for near real-time monitoring of E. coli in drinking water samples. Furthermore, this system can be applied independently or in conjunction with other methods as a part of an array of biochemical assays in order to reliably detect E. coli in water. / Dissertation/Thesis / Doctoral Dissertation Civil and Environmental Engineering 2015

Rapid detection of foodborne pathogen is one of the most urgent problems in the world, because foodborne pathogen could cause serious illness, such as nausea, vomiting and diarrhea. We have developed a sensitive microfluidic system based on dendrimers and aptamers for rapid detection of Escherichia coli O157:H7 at very low cells concentration. Dendrimers, with high level of functional groups and homogeneous spherical shape, are prefect nanoscale polymers used as a template material by increasing sensitivity and specificity of analytes detection in microfluidics. In this work, we develop a sensitive microfluidic system based on dendrimers and aptamers for detecting Escherichia coli O157:H7 at very low cell concentrations. Carboxyl functionalized G7-polyamidoamine (PAMAM-COOH) dendrimers are immobilized on (3-aminopropyl)-trimethoxysilane (APTMS) pretreated microfluidic channels. The aptamers are subsequently conjugated on the immobilized dendrimes through chemicals. The sensitivity and specificity are validated by injecting fluorescein isothiocyanate (FITC) labelled Escherichia coli O157:H7 at various cells concentration into the resulting microchannels, indicating that the detectable cells concentration can be reached as low as 100 (cells/ml) and the detection time is 10 hours. To further exploit and improve the work efficiency our microfluidic device, the microfluidic channel is designed into a staggered herringbone microchannel (SHM) to create the chaotic dynamics inside the microfluidic device, and the SHM is then simulated by a COMSOL software showing that the staggered herringbone structures can improve chaotic dynamics of designed microchannel and will enhance the probability of particles to attach on the surface of microdevice. All the results show that our approach has the potential to develop the field of rapid and accurate detection on foodborne pathogens.

Documentation exists that mothers can pass the cariogenic bacteria Streptococcus mutans to their infants. The newest technology to identify Streptococcus mutans is a rapid detection saliva test. Two hundred patients above the age of 18 were targeted using random selection in a Louisville, Kentucky dental office. Patients signed an informed consent form and were given a qualifying questionnaire. Patients received 2 bitewing x-rays and a charted DMFT index and were administered the saliva test. While the null hypothesis was rejected using the chi square test, the results were inconclusive due to expected values. However, other chi square results revealed that the test worked or had the potential to work. Furthermore, it was concluded that the test had high specificity. Further research is warranted; however, the saliva test in combination with the DMFT and x-rays are instrumental tools for the dental professional in educating patients and prevention.

The goal of this project was to incorporate bacteriophage with Bioluminescent Assay in Real-Time Loop-mediated isothermal amplification (BART-LAMP) for the rapid detection of Mycobacterium avium subspecies paratuberculosis (MAP). As the causative agent of Johne’s Disease, there are no rapid detection methods that are suitable in specificity and sensitivity. A screening assay for phage isolation was developed, and over 400 samples were screened for the isolation of a bacteriophage against MAP. One novel Mycobacterium phage was isolated and characterized using transmission electron miscroscopy, host range studies, restriction enzyme digestion, and pH and temperature stability. It was sequenced, annotated, and underwent an in silico protein analysis. No pathogenic or lysogenic genes were detected, and it was found to be related to Gordonia phage GTE2. BART-LAMP was applied to the detection of the isolated phage using purely extracted DNA and crude phage lysate, showing that phages could be detected successfully. / Beef Cattle Research Council; Agriculture and AgriFood Canada through Growing Forward initiative