Hot Cell-Direct PCR Aimed at Specific Cell Detection

Since the polymerase chain reaction (PCR) was proposed, it has become an essential method in the field of biological gene analysis, providing a method to amplify DNA sequences of interest. To detect and/or analyze genes in cells, the gene or expressed gene must first be extracted before PCR. This procedure takes time and may result in the loss of samples. In order to avoid such drawbacks, two methods, hot cell-direct PCR and reverse transcription-PCR (RT-PCR), were invented, to detect genes in cells. Using hot cell-direct PCR, specific genes in microbial cells such as invA in Salmonella enterica have been easily detected and applied to discriminate Archaea from bacteria. As hot cell-direct PCR and RT-PCR are fairly simple processes, they can be applied to detect genes in single cells. We developed an original compact disc (CD)-shaped microfluidic device with microchambers for single-cell isolation and a detection system for expressed genes in isolated single cells in a microchamber on the device. We succeeded in the detection of PCR and RT-PCR products in individual cells and successfully detected S. enterica cells by hot cell-direct PCR. Expressed genes in Jurkat cells—human leukemia T cells—were analyzed by this method

Development and evaluation of molecular-based assays for detecting Salmonella serovars in various food commodities

As a leading cause of foodborne illnesses and outbreaks, Salmonella poses a major public health risk in the United States and worldwide. Various food commodities including meat and poultry, eggs, and fresh produce can serve as the transmission vehicles for Salmonella infections. To better ensure the safety of these products and protect public health, rapid, accurate, and reliable detection methods for Salmonella are needed. Molecular-based methods like loop-mediated isothermal amplification (LAMP), have gained wide applications in Salmonella detection, owning to their rapidity, specificity, and sensitivity. However, there is a paucity of data on the robustness of these assays. And very recently, bioluminescence assay in real-time (BART) was used as a new and effective platform to detect LAMP products, and this combination has not been evaluated before. This dissertation research evaluated the robustness of two LAMP assays in comparison with PCR, examined the application of LAMP assays in detecting Salmonella specifically in food items, and developed a novel LAMP-BART assay for Salmonella detection. The LAMP assays achieved robust detection of Salmonella under abusive preparation and running temperatures, also demonstrated greater tolerance than PCR to various inhibitors. They achieved 100% accuracy among 185 strains. The limits of detection of LAMP for Salmonella strains belonging to ten serovars were 1 to 10 cells per reaction in pure culture, 100-fold more sensitive than PCR. In spiked egg homogenates, it could detect Salmonella serovars Enteritidis and Typhimurium down to 10^4 CFU/25 ml egg homogenates directly and 1 CFU/25 ml with 8 h enrichment. In spiked produce (cantaloupe, jalapeno pepper, tomatoes, sprouts, and lettuces), the detection limits ranged from 10^4 to 10^6 CFU/25 g produce, which were comparable to qPCR. Coupled with 6 to 8 h of enrichment, LAMP consistently detected in produce samples spiked with very low levels of Salmonella cells, with the exception of sprouts. Based on these evaluations and further development, LAMP demonstrated to be a rapid and robust alternative to PCR-based assays for Salmonella detection and could be adopted by food industries and regulatory agencies in routine product testing for Salmonella to improve product safety and protect public health

Development of diagnostic platform for detection of biological agents and toxic microalgae using isothermal amplification

L'objectiu principal d’aquesta tesis és desenvolupar sensors d’ADN portàtils i fàcils d’utilitzar per analitzar material genètic al lloc on sigui necessari, superant les limitacions de les tecnologies actuals. Per dur-ho a terme, els sensors d’ADN s’han dissenyat integrant en un únic dispositiu dues tecnologies: l’amplificació isotèrmica d’ADN, tant en fase líquida com en fase sòlida, i la tecnologia de microarrays. A la tesis es detallen els resultats obtinguts per assolir els objectius específics, que inclouen: el desenvolupament d’una plataforma d’anàlisi d’ADN en fase líquida per a la detecció simultània de diversos organismes. L’ús d’amplificació isotèrmica d’ADN en superfície (fase sòlida), combinant tant amb detecció òptica com detecció electroquímica per a la detecció de mostres reals. L’estudi i optimització de la química de superfície dels sensors d’ADN en fase sòlida, i finalment, l’amplificació isotèrmica d’ADN d’algues tòxiques emprant dNTPs modificats amb molècules redox, fet que permet mesurar directament l’ADN amplificat reduint el temps total d’anàlisi.El objetivo principal de la tesis es desarrollar sensores de ADN portátiles y fáciles de utilizar para analizar material genético allí donde sea requerido, superando las limitaciones de las tecnologías actuales. Para llevarlo a cabo, los sensores de ADN se han diseñado integrando en un único dispositivo dos tecnologías: la amplificación isotérmica de ADN tanto en fase líquida como en fase sólida, y la tecnología de microarrays. En la tesis se detallan los resultados obtenidos para lograr los objetivos específicos, que incluyen: el desarrollo de una plataforma de análisis de ADN en fase líquida para la detección simultánea de distintos organismos. El uso de amplificación isotérmica de ADN en superficie (fase sólida) combinado tanto con detección óptica como con detección electroquímica para la detección de muestras reales. El estudio y optimización de la química de superficie de los sensores de ADN en fase sólida, y finalmente, la amplificación isotérmica de ADN de algas tóxicas utilizando dNTPs modificados con moléculas redox, cosa que permite medir el ADN amplificado directamente, reduciendo así el tiempo tota de análisis.The main goal of this doctoral thesis is to present alternative approaches in the field of DNA biosensors, design and develop new isothermal amplification protocols compatible with a portable, easy-to use device that can be deployed for analysis of genetic material at the point-of-care/need, while overcoming some of the currently existing limitations. In order to achieve this objective, a variety of different strategies of effective solid-phase immobilisation and liquid-phase and solid-phase isothermal enzymatic amplification have been studied to achieve lower detection limits with rapid and easy to carry out assays. This work presents a convenient, flexible solution for detecting DNA with biosensors, exploiting a general concept of liquid-phase and solid-phase isothermal amplification and detection, thus integrating two nucleic acid tests, PCR and microarrays, in one single device. The thesis report the work performed to achieve the specific objectives of this doctoral thesis: the development of multiplexing platform for simultaneous detection of several targets, the use of solid-phase recombinase polymerase amplification strategy for DNA amplification with optical and electrochemical detection, the detection of real samples, the study of the surface chemistry and the combination of isothermal amplification with redox labelled dNTPs for the amplification and detection of toxic microalgae as an innovative method that permits incorporation of labels throughout the amplification process facilitating direct electrochemical detection of the DNA products and an inherent shortening of assay time