Intelligent Real-Time Polymerase Chain Reaction System with Integrated Nucleic Acid Extraction for Point-of-Care Medical Diagnostics

Date of Award

2023

Degree Name

Ph.D. in Electrical Engineering

Department

Department of Electrical and Computer Engineering

Advisor/Chair

Yvonne Sun

Abstract

The recent pandemic of Corona-virus Disease 2019 (COVID-19) caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) showed an urgent need to rapidly and accurately identify the genetic material of SARS-CoV-2, an enveloped ribonucleic acid (RNA) virus, in upper respiratory specimens from people. Further, foodborne and waterborne diseases are not only spreading faster, but also appear to be emerging more rapidly than ever before and are able to circumvent conventional control measures. The Polymerase Chain Reaction (PCR) system is a well-known diagnostic tool for many applications in medical diagnostics, environmental monitoring, and food and water quality assessment. Here, we describe the design, development, and testing of a portable, low-cost, and real-time PCR system that can be used in emergency health crises and resource-poor situations. The described PCR system incorporates real-time reaction monitoring using fluorescence as an alternative to gel electrophoresis for reaction analysis, further decreasing the need of multiple reagents, reducing sample testing cost, and reducing sample analysis time. The bill of materials cost of the described system is approximately $340. The described PCR system utilizes a novel progressive selective proportional–integral–derivative controller that helps in reducing sample analysis time. In addition, the system employs a novel primer-based approach to quantify the initial target amplicon concentration, making it well-suited for food and water quality assessment. The developed PCR system performed DNA amplification at a level and speed comparable to larger and more expensive commercial table-top systems. The fluorescence detection sensitivity was also tested to be at the same level as commercially available multi-mode optical readers, thus making the PCR system an attractive solution for medical point-of-care and food and water quality assessment. In general, sensitive testing methods require genetic material extraction from the specimen. Unfortunately, current commercially available extraction kits are expensive and involve time-consuming and laborious extraction procedures. To overcome the difficulties associated with common extraction methods, we propose a simple enzymatic assay for the nucleic acid extraction step using heat mediation to improve the PCR reaction sensitivity. Our protocol was tested on Human Coronavirus 229E (HCoV-229E) as an example, which comes from the large coronaviridae family of viruses that affect birds, amphibians, and mammals, of which SARS-CoV-2 is a member. The proposed assay was performed using a low-cost, custom-made, real-time PCR system that incorporates thermal cycling and fluorescence detection. It had fully customizable reaction settings to allow versatile biological sample testing for various applications, including point-of-care medical diagnosis, food and water quality testing, and emergency health situations. Our results show that heat-mediated RNA extraction is a viable extraction method when compared to commercial extraction kits. Further, our study showed that extraction has a direct impact on purified laboratory samples of HCoV-229E, but no direct impact on infected human cells. This is clinically relevant, as it allows us to circumvent the extraction step on clinical samples when using PCR. Last, traditional testing methods require some external expertise to interpret reaction results. This is a limiting factor to ubiquitous medical testing since low-resource areas may lack such expertise or personnel. The use of Artificial Neural Networks (ANN) for automatic PCR result analysis was demonstrated on low-power microcontroller and incorporated into the proposed system. This is useful for full system autonomy when employing PCR for low-resource point-of-care testing.

Keywords

polymerase chain reaction (PCR), fluorescence sensing, real-time PCR, RNA extraction, point-of-care diagnostics, COVID-19, food and water quality, low-cost PCR, proportional–integral–derivative (PID) controller

Rights Statement

Copyright © 2023, Author

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