Optimization of Ultra-Fast Plasmonic RT-PCR for Detection of E. coli Water Contamination and Use in Point-of-Care Microbial Infection Diagnostics

Abstract

Since 1983, polymerase chain reaction (PCR) has been the method of choice for detection and diagnosis of infectious pathogens, due to its high specificity and selectivity. Reverse transcriptase, quantitative, PCR (RT-qPCR) is an especially relevant technique for detection of RNA-based microbials or genetically derived material. However, current methodologies and workflows are lengthy and require equipment that is regulated to central laboratories. Our laboratory has reinvented PCR, through the implementation and use of gold nanorods to develop a plasmonic PCR system. This has provided a platform to deliver rapid, in-situ testing of microbials in under 10 minutes, in a compact/portable system. The plasmonic PCR platform uses either fluorescence or label-free UV amplicon monitoring systems for diagnostic use. The goal of this project is to design and validate an RT-qPCR protocol on the plasmonic thermocycler, that can be specifically used for water quality testing. Using the Flic gene to target for amplification, RNA was isolated from several E. coli strains (TOP10, K12 and DH5a), and used to design a custom, RT-qPCR reagent kit for easy and efficient operation. Once optimized, the system can be applied as an easy-to-use, accessible, rapid technology for water quality monitoring of E.coli contamination in Indigenous communities. These RT-qPCR protocols can also be applied for point-of-care COVID-19 and HIV diagnostics. The results of the experiments thus far, have demonstrated comparable for plasmonic amplification vs. conventional PCR systems in around 13 minutes. Research is still underway to finalize the system for point-of-care diagnostics and in-situ water quality testing.

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McGill University is located on land which has long served as a site of meeting and exchange amongst Indigenous peoples, including the Haudenosaunee and Anishinabeg nations. McGill honours, recognizes and respects these nations as the traditional stewards of the lands and waters on which peoples of the world now gather. Today, this meeting place is still the home to many Indigenous people from across Turtle Island and we are grateful to have the opportunity to work on this land.

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