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Rolling Circle Amplification in Integrated Microsystems: An Uncut Gem toward Massively Multiplexed Pathogen Diagnostics and Genotyping
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab. Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, S-17165 Solna, Sweden.ORCID iD: 0000-0001-5958-5232
Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, S-17165 Solna, Sweden.;Indian Inst Technol Madras, Dept Biotechnol, Chennai 600036, Tamil Nadu, India..
Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, S-17165 Solna, Sweden..ORCID iD: 0000-0001-9985-0387
2021 (English)In: Accounts of Chemical Research, ISSN 0001-4842, E-ISSN 1520-4898, Vol. 54, no 21, p. 3979-3990Article, review/survey (Refereed) Published
Abstract [en]

CONSPECTUS: The 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 similar to 95 degrees 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 PLPRCA 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.

Place, publisher, year, edition, pages
American Chemical Society (ACS) , 2021. Vol. 54, no 21, p. 3979-3990
National Category
Biochemistry Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-305367DOI: 10.1021/acs.accounts.1c00438ISI: 000714106300001PubMedID: 34637281Scopus ID: 2-s2.0-85119129980OAI: oai:DiVA.org:kth-305367DiVA, id: diva2:1616527
Note

QC 20211203

Available from: 2021-12-03 Created: 2021-12-03 Last updated: 2025-02-20Bibliographically approved

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Soares, Ruben R. G.

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