| Abstract |
Food hygiene and safety are matters of significant importance to everyone. Annually, foodborne diseases are reported in a worldwide scale. Sources of the aforementioned diseases may be inappropriate handling during production, preparation, packaging or even food cooking. Although controls become increasingly stricter, cases are still reported due to ineffective food maintenance and handling. These phenomena are more intense to third world countries, where resources and the ability to follow the necessary regulations are not abundant. Apparently, low quality food has numerous consequences. The most important aspect is human and public health, since contaminated food may lead to more or less serious diseases, or even death. Moreover, disease spreading also poses a threat in case the contaminated food is commercially exported to other regions of the planet. The damage is not restricted to human lives though; it expands to overwhelming expenses for medical and clinical treatment, a very negative impact of the consumers’ trust towards the producer and can in consequence wreak havoc on the field’s economy and trading.
It is widely accepted that the vast majority of foodborne diseases is directly related to bacteria. The most important candidates are Clostridium perfringens, Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, Bacillus cereus, as well as members of the Salmonella genus. It is noteworthy however, that only about 20 bacteria species are responsible for most foodborne diseases, although that percentage is 90% or even higher.
As a result, to protect consumers’ health and avoid significant financial casualties, food safety control is imperative.
In the present work, we tried to detect the pathogen Salmonella enterica serovar Typhimurium, applying two different DNA amplification methods, the well-established PCR (Polymerase Chain Reaction), and the isothermal RCA (Rolling Circle Amplification). The aim was bacteria DNA amplification in milk samples, if it indeed existed. Detection was conducted using an acoustic SAW (Surface Acoustic Waves) Biosensor, which apart from its sensitivity and label-free detection potential, gives the option of combination with microfluidic systems to form integrated Lab-on-Chip (LoC) platforms. These platforms are expected to offer many features like speed and reduction in cost, testing of multiple samples in parallel, great detection sensitivity, avoiding the demand of expensive equipment and workspace, as well as the need of specialized and skilled personnel. Another advantage is that our method required no additional sample treatment, like bacteria culture, after the samples have been collected, reducing the time, cost and technical training conventional methods require.
Even though at this stage, in most cases, bacterial genomic DNA was used instead of actual bacteria, it is still possible to use them directly, after heating the sample to inflict thermal lysis. PCR and RCA were both tested for the amplification of a gene(s) of interest followed by direct detection with no further sample treatment like DNA clean-up. The results were promising as both amplification methods offered satisfactory detection limits (5cells/sample for PCR and 100cells/sample for RCA up to the current phase), implying our approach lacks nothing when compared to classic methodologies. The two methods were then compared in terms of simplicity, reagent and equipment requirements, speed, sensitivity etc. and then an effort of suggesting which of the two is more suitable to be used in Lab-on-Chip (LoC) platforms was made. It is important to note that during the time offered for a master diploma thesis (1 year), every possible effort was made to optimize the methods and especially RCA, in order to reach a conclusive protocol. Although the progress was satisfactory, there is still room for improvement. More time is needed for the method optimization, not only for its’ preparation and application, but also to conclude about its’ effectiveness (required time, product yield, detection limit, cost reduction etc.).
Throughout this thesis, I hope it will be made clear that application of rising technologies, such as microfluidic systems and acoustic biosensors, coupled with more traditional methods like PCR and RCA for the construction of LoC platforms, can offer many advantages, like increased sensitivity, cost-effective methods and parallel and rapid sample treatment for applications of great interest, such as diagnostics and food safety control.
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