Molecular methods for the detection of nucleic acids applied to infection and cancer diagnostics

There is now an array of molecular methods to detect, quantify and elucidate nucleic acids sequences (DNA & RNA) with the field in a constant state of developmental flux. Molecular approaches are now the standard for clinical viral diagnostics with the qPCR the cornerstone of most modern hospitals. However as shown during the Covid-19 pandemic, inherent complexity, costs and the need for advanced read-out thermocyclers impede its wide scale deployment across settings. There is an ongoing movement to develop novel diagnostic approaches which are cheaper and simpler to facilitate sensitive viral detection at scale. Such developments can enable the democratization of diagnostics, bringing us closer to POC and help ensure better preparedness for future pandemics. For most bacterial infections phenotypic based antimicrobial susceptible tests (AST) are used with some molecular assays which complement them. However, the majority of these clinical tests require a number of days to provide actionable results. Such delays impact patient outcomes and promote imprecise antibiotic use. With the growing threat of antimicrobial resistance (AMR) there is an obvious need for novel rapid tests which accelerate clinical decisions and promote antibiotic stewardship. Sequencing based approaches have also recently become more accessible to clinical and research-based microbiology laboratories. They can provide insights into mechanisms of antimicrobial resistance and help track variant evolution. This revolution in high throughput sequencing technologies has also seen the concurrent development of numerous novel methods applied to human health and disease. These methods are now increasingly important to help identify high risk patients and inform cancer treatment. In this thesis the role of molecular diagnostics in microbial detection are reviewed along with an overview of phenotypic - antimicrobial susceptibility tests (AST) and their recent advances. In addition, I present an overview of sequencing technology and its application to the detection of rare DNA variants.

In Paper I, we developed a method to detect SARS-CoV-2 in a reverse transcription loop mediated isothermal amplification reaction (RT-LAMP) using in-house produced (non-commercial) enzymes directly on heat inactivated (non-extracted) samples. This work was developed during the Covid-19 pandemic as a potential alternative for viral diagnostics. We aimed to complement the gold standard qPCR which was unable in certain cases to fulfill the testing needs. We focused on developing an isothermal approach using in house produced enzymes without the need for ribonucleic acid (RNA) extraction or heat cycling devices to enable a wider potential usage. We produced and isolated reverse transcriptase's and strand displacing DNA polymerases, tested and optimized their performance and subsequently benchmarked our approach against commercial alternative showing comparable results. We then validated our method in heat inactivated Covid-19 patient samples which had been diagnosed using qPCR.

In Paper II, we applied the RT-LAMP reaction into an integrated standalone centrifugal and heating platform with POC diagnostics in mind. Here, we incorporated discs packed with n-benzyl-n-methylethanolamine (NBNM) modified agarose beads. These beads enabled the removal of primer dimers and allowed an enhanced end-point fluorescent detection of the sample via a smartphone at room temperature. We subsequently validated the approach in a range of Covid-19 clinical samples, demonstrating a detection limit of 100 RNA copies within one hour on non-extracted samples. Thus providing an alternative approach to enable cheap mass scale testing in resource-limited environments or closer to POC diagnostics.

In Paper III, we developed a protocol to produce DNA Nanoball structures during an RT-LAMP via the addition of compaction oligos. These structures are produced in solution of approximately one micron in size without the need for any nucleation agents e.g. microbeads. We were then able to quantify these nanoballs individually in an impedance based microfluidic chamber without the need for any labelling (e.g. colorimetric or fluorescence). We initially developed our protocol using synthetic SARS-CoV-2 RNA. We then validated our ability to produce DNA Nanoballs from heat inactivated Covid-19 clinical samples. We thereafter produced Nanoballs from different target sequences and detected them with impedance. These included for HIV, influenza, mycobacterium and the AMR gene beta-lactamase highlighting the flexibility of our approach.

In Paper IV we developed a novel approach to produce double stranded (ds) unique molecular identifiers (dsUMIs) within a standard short read sequencing library preparation workflow. This enables the accurate detection of low frequency variants in DNA molecules which are relevant in many settings including cancer (e.g. minimal residual disease). In standard workflows the detection of such rare variants is complicated by errors which can occur at a similar frequency. One solution to address this is to label both strands of a DNA molecule with a barcode (dsUMI). This can then allow for the identification and removal of a number of low frequency errors and resolution of boundary collisions. However, to develop such dsUMI barcodes is laborious and expensive. Here our approach called one pot double stranded UMI sequencing (OPUSeq) produces a dsUMI DNA molecule within a modified PCR. We then show its ability to identify rare variants in an experiment which mimics minimal residual disease (MRD). Our research also revealed that the fragmentase mix produces low frequency errors on both strands across the molecule.

In our Preliminary data, we developed a rapid one pot multiplex reaction. Here we use molecular inversion probes coupled to sequencing based readout to detect bacterial and fungal targets, as well as AMR genes. We optimized the method using ZymoBiomics microbiome cell (Bacterial and Fungi) standards and synthetic fungal ITS and AMR spike-ins. Currently we are validating our approach to detect infections from blood samples taken from acute myeloid leukemia (AML) patients.

List of scientific papers

I. Direct detection of SARS-CoV-2 using non-commerical RT- LAMP reagents on heat-inactivated samples. Alisa Alekseenko*, Donal Barrett*, Yerma Pareja-Sánchez*, Rebecca J Howard, Emilia Strandback, Henry Ampah-Korsah, Urška Rovšnik, Silvia Zuniga-Veliz, Alexander Klenov, Jayshna Malloo, Shenglong Ye, Xiyang Liu, Björn Reinius, Simon J Elsässer, Tomas Nyman, Gustaf Sandh, Xiushan Yin, Vicent Pelechano. Scientific Reports 2021, Jan 19;11(1):1820. https://doi.org/10.1038/s41598-020-80352-8
*Authors contributed equally to this study

II. Sample-to-answer COVID-19 nucleic acid testing using low- cost centrifugal microfluidic platform with bead-based signal enhancement and smartphone read-out. Ruben R G Soares, Ahmad S Akhtar, Ines F Pinto, Noa Lapins, Donal Barrett, Gustaf Sandh, Xiushan Yin, Vicent. Pelechano, Aman Russom. Lab Chip, 2021, 21, 2932.
https://doi.org/10.1039/D1LC00266J

III. Digital Assay for rapid electronic quantification of clinical pathogens using DNA Nanoballs. Muhammad Tayyab*, Donal Barrett*, Gijs van Riel, Shujing Liu, Björn Reinius, Curt Scharfe, Peter Griffin, Lars M. Steinmetz, Mehdi Javanmard, Vicent Pelechano. Sci. Adv.9,eadi4997(2023). https://doi.org/10.1126/sciadv.adi4997
*Authors contributed equally to this study

IV. OPUSeq simplifies detection of low frequency DNA variants and uncovers fragmentase-associated artifacts. Alisa Alekseenko, Jingwen Wang, Donal Barrett, Vicent Pelechano. NAR Genomics and Bioinformatics 2022, Jun 27;4(2):lqac048. https://doi.org/10.1093/nargab/lqac048