Paper-Based Extraction and Amplification of Bacterial Genomic DNA to Monitor Food Safety

Abstract

The development of rapid detection methods for bacterial contamination in the industrial agriculture sector is vital for improving food safety and public health. Furthermore, it is important to tailor these rapid methods for low-resource settings, because the majority of foodborne illness outbreaks occur in developing nations. Currently, the most widely used methods rely on nucleic acid testing using the polymerase chain reaction (PCR). This detection reaction provides repeatable results, is highly sensitive, and is highly specific, as it can detect a single strain within a species. However, PCR is reliant on proper sample pre-treatment to remove inhibitory contaminants which can affect downstream results, which leads to a trade-off between detection time and sensitivity of results. Rolling circle amplification (RCA) is another potential detection reaction which has the same advantages and is also better suited to low-resource settings, as it works at room temperature. This thesis reports on the development of a rapid sample preparation method that can be seamlessly integrated into simple PCR and is also well suited to low-resource settings due to the low cost and high availability of the required reagents. A modification of the hot sodium hydroxide plus tris (HotSHOT) lysis reaction was implemented to extract genomic DNA (gDNA), which was then captured onto cellulose filter paper, allowing for multiple samples to be simultaneously processed in under 30 minutes. This pre-treatment can even recover gDNA for detection from samples that would have caused complete inhibition of PCR. The calculated limit of detection (LoD) of extraction followed by simple PCR was similar to that of government-approved commercial kits, without needing a lengthy bacterial enrichment step. Improvements are needed to make this a truly quantitative detection system. Finally, our paper-based pre-treatment was integrated into an RCA reaction to detect at least 105 cells, which provides proof-of-concept for combining paper-based sample preparation with isothermal amplification of target nucleic acids.