Point-of-Care Nucleic Acid Test Platforms for the Direct Detection of RNA and DNA

Abstract

Bio-analytical device platforms based on passive fluid manipulation and isothermal nucleic acid amplification technology are versatile and expected to transform healthcare practice by rapidly detecting disease, guiding treatment at a low cost, and making it accessible to all. Paper wicking and fluid flow in channels empowered by capillary force and appropriately integrated with detection chemistry and devices are best suited for point-of-care (POCT) and at-home diagnostics. Loop-mediated isothermal amplification (LAMP) and lateral flow dipstick (LFD) have been developed for years and have advanced significantly in the COVID-19 pandemic era. However, several issues remain unresolved, hindering the realization of the full potential of these technologies as cutting-edge POCT technology. In addition to the limited tools and methods for developing an optimal and hybrid LAMP-LFD method, the grand challenge is to control passive and dynamic fluid flow to have the desired analytical performance for "sample-in-result-out" nucleic acid detection. Achieving a multi-parameter standard for an ideal POCT acronymized "ASSURED" (affordability, sensitivity, specificity, user-friendliness, reliability, equipment-freeness, and deliverability) is almost impossible, but has been diligently followed in the current development of methods and systems beneficial in the design of portable analytical devices for the direct detection of DNA and RNA in crude clinical samples.

COVID-19 is an emerging disease caused by a novel coronavirus (SARS-CoV-2) that has escalated into a full-blown pandemic within a short time. The virus's high contagiousness has caused health concerns and devastating economic and social disruption. It is indisputable that simple and affordable screening tests in the vicinity of demand and accessible to all are crucial. This is necessary not only to combat the emerging (and now endemic) SARS-CoV-2 but also to address the recently re-emerging monkeypox virus (MPXV), which has rapidly spread into countries where it was not present before. Furthermore, there are diseases, such as cancer induced by HPV, which exhibit a disproportionate and severe impact on individuals with restricted access to diagnostic resources. Therefore, while our primary focus remains on the development of assays and chemistry platforms, a novel LAMP-LFD assay was simultaneously developed to address these public health-significant zoonotic and oncogenic infectious diseases. For the SARS-CoV 2 LAMP-LFD assay, the analytical performance was evaluated using a panel of controls and at a multi-center clinical trial site with more than 97% overall accuracy after RNA extraction and up to 85% without RNA extraction using samples collected in Virus Transport Medium (VTM). To further address the issue of deliverability to, and application by end-users, a stable and long-lasting dry chemistry format was prepared and multiplexed with other gene targets or coupled with a sample internal control. This modification in the chemistry is indispensable for enabling effortless transportation, prolonged shelf storage, and the application of kits in non-laboratory environments. Integrating the chemistry with the microfluidic module and handheld device enabled molecular screening of SARS-CoV-2 RNA at the point of demand, including at-home screening. Once the prototype is validated in laboratory and clinical trial settings, we are certain that the POCT device can provide a portable and safe-to-operate molecular diagnostics alternative for public health.

One of the issues addressed in LAMP-LFD assay development was selecting the preferred hapten tag location. Current practices are identified as suboptimal, as reported methods fail to mention comprehensive and systematic screening of essential combinations of hapten-tagged primer sets. Preferred tag locations were identified by studying a set of de novo-designed and literature-modified LAMP primer sets using an orthogonal design and adhering to 'ASSURED' guided standards in POCT. To identify possible patterns related to tag location, amplification speed, and performance on the LFD strip, an analysis was conducted. This led to the establishment of five different tagging modes (Mode A to Mode E) according to the specific roles of the tagged primers in the LAMP reaction. These modes were instrumental in formulating principles for the design of the LAMP-LFD assay. Interestingly, hapten modification caused some LAMP-LFD primer sets to have superior amplification speed compared to their unmodified counterparts, signifying the importance of orthogonal design and experimentation before deciding the final optimal LAMP-LFD primer sets. Moreover, during the development of single-plex LAMP-LFD assays, incorporating additional and in-LAMP-tagged primers from the pools of probes or primers used in sequence-specific nucleic acid detection methods like qPCR and hybridization assays proved to be effective in developing highly specific LAMP-LFD assays. Our suggested approach in primer design can serve as a fundamental strategy for mitigating the inherent issue of spurious amplification in LAMP technology.

The challenge in standard LAMP assay development lies in the requirement for six primers, which can be a complex and demanding task. Moreover, performing LAMP multiplexing, which has double or triple the primer amount, further increases the design complexity. There is neither software nor an effective method that helps to develop a complex assay for multiple target detection in a single tube reaction using LAMP. The difficulty in developing a multiplex LAMP-LFD assay is further compounded by the need to address tag selection and optimization for multiple targets. Hence, a new workflow is introduced involving three layers of optimization: 1) Development of a singleplex LAMP-LFD assay using the orthogonal screening method; 2) refinement of the multiplex primer mix ratio guided by amplification kinetics, and 3) optimization of the tagged primer concentration guided by the intensity of color bands on the LFD at the respective tag locations. The effectiveness of the workflow in developing an intricate LAMP assay was demonstrated through the successful creation of various LAMP duplex and triplex assays. These assays included detection using a lateral flow assay, highlighting the workflow's utility as a platform for the development of LAMP-LFD molecular assays for point-of-care testing. Our method has been verified by successfully detecting clinical samples in both duplex and triplex assays developed based on the workflow.

Dry chemistry is an essential requirement for successfully applying the LAMP-LFD assay in POCT. This is due to the requirements of easy transportation, stable storage, and operability in a minimally controlled environment for POCT. However, dry chemistry assay development using an enzyme source containing glycerol as a stabilizer, such as in a commercial LAMP enzyme master mix, is challenging because glycerol affects the freeze-drying process and needs to be removed before drying. To address this, a protocol was developed to treat a commercially available enzyme master mix in a high-glycerol medium and customize it into a shelf-stable, ready-to-go dry master mix. The protocol includes steps for depleting glycerol and concentrating enzymes in a simple glycerol exchange buffer. Subsequently, the core-enzymatic reaction components, an optimized LAMP-LFD primer mix in a buffer, and freeze-dry stabilizers are re-supplemented. This is followed by a controlled freeze-drying process, after which the tubes are packed in an inert environment. This protocol was successfully utilized to develop a ready-to-go dry LAMP-LFD chemistry for SARS-CoV-2.

A significant breakthrough and revelation was the development of a novel one-pot pathogen detection system based on LAMP. This system integrates an exceptionally specific assay to identify true positive samples even before the entire LAMP reaction process is concluded. The system shortens the reaction duration and reduces the likelihood of false positive results due to non-specific amplification or signal error (e.g., the sample has a too high pH) in the LAMP reaction. The invention integrates an immunochromatographic assay, such as a lateral flow assay (LFA), and colorimetric, fluorometric, or both to track the LAMP reaction and enable real-time pathogen detection, thereby improving the efficiency thereof with high specificity. This distinctive approach empowers our platform to detect the target with shorter incubation times, under a constant temperature. It also offers improved specificity, the potential for semi-quantitative analysis, and supports various result interpretations within the sample. The concept was effectively employed for the real-time detection of SARS-CoV-2 and MPXV in a lateral flow assay (LFA) format, and it was also suggested for integration into a smart and portable device designed for molecular testing in at-home settings.

Finally, preliminary results and future work on a novel device platform were introduced for a complete solution towards a "sample-in-result-out" POCT and at-home use molecular device platform. These include preliminary results on a disposable microfluidic module for contamination-free and closed amplification and a simple electronic device that helps run isothermal amplification. The resulting prototype is a disposable microfluidic module with multiple reaction chambers loaded with dry reagents and filled with extraction buffer, designed to achieve “sample-in-result-out” with the electronic device to provide an operational guide to the user and automatically monitor the reaction. Moreover, mobile phone software was drafted to display the device's status, helps to operate real-time LAMP-LFD and interact with the user through the step-by-step operation of the selected diagnostic mode in a user-friendly format.

Date of Award	12 Sept 2023
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	M YANG (Supervisor)