Abstract
ConspectusThe development of robust methods allowing the precise detection of specific nucleic acid sequences is of major societal relevance, paving the way for significant advances in biotechnology and biomedical engineering. These range from a better understanding of human disease at a molecular level, allowing the discovery and development of novel biopharmaceuticals and vaccines, to the improvement of biotechnological processes providing improved food quality and safety, efficient green fuels, and smart textiles. Among these applications, the significance of pathogen diagnostics as the main focus of this Account has become particularly clear during the recent SARS-CoV-2 pandemic. In this context, while RT-PCR is the gold standard method for unambiguous detection of genetic material from pathogens, other isothermal amplification alternatives circumventing rapid heating–cooling cycles up to ∼95 °C are appealing to facilitate the translation of the assay into point-of-care (PoC) analytical platforms. Furthermore, the possibility of routinely multiplexing the detection of tens to hundreds of target sequences with single base pair specificity, currently not met by state-of-the-art methods available in clinical laboratories, would be instrumental along the path to tackle emergent viral variants and antimicrobial resistance genes. Here, we advocate that padlock probes (PLPs), first reported by Nilsson et al. in 1994, coupled with rolling circle amplification (RCA), termed here as PLP-RCA, is an underexploited technology in current arena of isothermal nucleic acid amplification tests (NAATs) providing an unprecedented degree of multiplexing, specificity, versatility, and amenability to integration in miniaturized PoC platforms. Furthermore, the intrinsically digital amplification of PLP-RCA retains spatial information and opens new avenues in the exploration of pathogenesis with spatial multiomics analysis of infected cells and tissue.The Account starts by introducing PLP-RCA in a nutshell focusing individually on the three main assay steps, namely, (1) PLP design and ligation mechanism, (2) RCA after probe ligation, and (3) detection of the RCA products. Each subject is touched upon succinctly but with sufficient detail for the reader to appreciate some assay intricacies and degree of versatility depending on the analytical challenge at hand. After familiarizing the reader with the method, we discuss specific examples of research in our group and others using PLP-RCA for viral, bacterial, and fungal diagnostics in a variety of clinical contexts, including the genotyping of antibiotic resistance genes and viral subtyping. Then, we dissect key developments in the miniaturization and integration of PLP-RCA to minimize user input, maximize analysis throughput, and expedite the time to results, ultimately aiming at PoC applications. These developments include molecular enrichment for maximum sensitivity, spatial arrays to maximize analytical throughput, automation of liquid handling to streamline the analytical workflow in miniaturized devices, and seamless integration of signal transduction to translate RCA product titers (and ideally spatial information) into a readable output. Finally, we position PLP-RCA in the current landscape of NAATs and furnish a systematic Strengths, Weaknesses, Opportunities and Threats analysis to shine light upon unpolished edges to uncover the gem with potential for ubiquitous, precise, and unbiased pathogen diagnostics.
Highlights
Required for multiple rounds of strand denaturation, primer annealing, and polymerization limit the matrix compatibility and bioanalytical versatility of polymerase chain reaction (PCR). These technical challenges motivated the development of isothermal amplification techniques allowing linear or exponential amplification at a constant and preferably lower temperature. Techniques such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) resort to polymerase enzymes with high strand displacement activity avoiding the need for a denaturation or high temperature polymerization step and/or recombinase enzymes combined with singlestranded DNA binding proteins, allowing efficient primer hybridization without temperature-driven denaturation and annealing steps.[5]
We have extensively demonstrated the applications of a single round of both rolling circle amplification (RCA) and C2CA for the sensitive, specific, and multiplex detection of a wide range of viral pathogens.[2,38−49] In the specific context of RNA viruses, RCA has been used for the detection of Zika Virus,[48−50] hypervariable viruses including NDV,[46] retroviruses such as HIV,[47,51] hemorrhagic fever causing viruses such as Crimean Congo Hemorrhagic Fever Virus (CCHFV) and Ebola,[48,52] influenza,[2,53,54] and coronaviruses such as SARS55 responsible for the 2003 epidemic as well as SARS-CoV-256−58 causing the current pandemic
The high degree of multiplexing in the range of tens to hundreds of targets combined with high specificity stands out as unique features with the potential to improve targeted diagnostics without the inherent complexity of nextgeneration sequencing (NGS) methods
Summary
Ever since the advent of polymerase chain reaction (PCR), the means of achieving highly sensitive and precise amplification of nucleic acid sequences have paved the way toward remarkable breakthroughs in biotechnology and biomedical engineering. The intrinsic heating−cooling cycles up to ∼95 °C required for multiple rounds of strand denaturation, primer annealing, and polymerization limit the matrix compatibility and bioanalytical versatility of PCR. These technical challenges motivated the development of isothermal amplification techniques allowing linear or exponential amplification at a constant and preferably lower temperature. Techniques such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) resort to polymerase enzymes with high strand displacement activity avoiding the need for a denaturation or high temperature polymerization step and/or recombinase enzymes combined with singlestranded DNA binding proteins, allowing efficient primer hybridization without temperature-driven denaturation and annealing steps.[5] These, along with other isothermal amplification strategies,[5] generate either short clonal amplicons with few hundreds of base pairs or nonclonal long dsDNA amplicons. PLPs, explored in detail in the subsequent section, provide the adequate boost in specificity to RCA, often lacking specificity when resorting exclusively to circle hybridization
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