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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 ∼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.

Isothermal amplification techniques are emerging nowadays for the rapid and accurate detection of pathogenic bacteria in low resource settings, where many infectious diseases are endemic, and the lack of reliable power supply, trained personnel and specialized facilities pose critical barriers for timely diagnosis. This work addresses the detection of E. coli based on DNA isothermal amplification performed on magnetic particles (MPs) followed by electrochemical genosensing on disposable electrodes by square-wave voltammetry. In this approach, the bacterial DNA is preconcentrated using a target-specific magnetic probe and then amplified on the MPs by rolling circle amplification (RCA). Two different electrochemical readout methods for the RCA amplicons are tested. The first one relied on the labelling of the magnetic RCA product with a digoxigenin probe followed by the incubation with antiDIG-HRP antibody as electrochemical reporter. In the second case, the direct detection with an HRP-probe was performed. This latter strategy showed an improved analytical performance, while simultaneously avoiding the use of thermocyclers or bulky bench top equipment.

Changes in cell identities and positions underlie tissue development and disease progression. Although single-cell mRNA sequencing (scRNA-Seq) methods rapidly generate extensive lists of cell states, spatially resolved single-cell mapping presents a challenging task. We developed SCRINSHOT (Single-Cell Resolution IN Situ Hybridization On Tissues), a sensitive, multiplex RNA mapping approach. Direct hybridization of padlock probes on mRNA is followed by circularization with SplintR ligase and rolling circle amplification (RCA) of the hybridized padlock probes. Sequential detection of RCA-products using fluorophore-labeled oligonucleotides profiles thousands of cells in tissue sections. We evaluated SCRINSHOT specificity and sensitivity on murine and human organs. SCRINSHOT quantification of marker gene expression shows high correlation with published scRNA-Seq data over a broad range of gene expression levels. We demonstrate the utility of SCRINSHOT by mapping the locations of abundant and rare cell types along the murine airways. The amenability, multiplexity, and quantitative qualities of SCRINSHOT facilitate single-cell mRNA profiling of cell-state alterations in tissues under a variety of native and experimental conditions., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: MN and XQ hold shares in CARTANA AB, a company that commercializes in situ sequencing technology.

Emerging tropical viruses have caused serious outbreaks during the recent years, such as Ebola virus (EBOV) in 2014 and the most recent in 2018 to 2019 in Congo. Thus, immediate diagnostic attention is demanded at the point of care in resource-limited settings, because the performance and the operational parameters of conventional EBOV testing are limited. Especially, their sensitivity, specificity, and coverage of other tropical disease viruses make them unsuitable for diagnostic at the point of care. Here, a padlock probe (PLP)-based rolling circle amplification (RCA) method for the detection of EBOV is presented. For this, a set of PLPs, separately targeting the viral RNA and complementary RNA of all seven EBOV genes, was used in the RCA assay and validated on virus isolates from cell culture. The assay was then translated for testing clinical samples, and simultaneous detection of both EBOV RNA types was demonstrated. For increased sensitivity, the RCA products were enriched on a simple and pump-free microfluidic chip. Because PLPs and RCA are inherently multiplexable, we demonstrate the extension of the probe panel for the simultaneous detection of the tropical viruses Ebola, Zika, and Dengue. The demonstrated high specificity, sensitivity, and multiplexing capability in combination with the digital quantification rendered the assay a promising diagnostic tool toward tropical virus detection at the point of care., (Copyright © 2020 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Recent advances of image-based in situ mRNA quantification methods allow to visualize where in a tissue section a set of genes is expressed. It enables to map large numbers of genes in parallel and by capturing cellular boundaries allows to assign genes to cells. Here, we present a high-throughput, multi-targeted gene expression profiling technique called in situ sequencing that is capable of localizing hundreds of genes simultaneously and supports cell type classifications that follow transcriptome-based taxonomy. In situ sequencing is a targeted, amplified, and barcoded approach using padlock probes (PLPs) and rolling circle amplification (RCA). The current protocol relies on mRNA fixation, mRNA reverse transcription, residual mRNA degradation, and PLP hybridization. PLPs are amplified by RCA and labeled with fluorophore-conjugated probes, allowing their detection under conventional fluorescence microscopes.

Infection by DNA viruses such as human adenoviruses (HAdVs) causes a high-level accumulation of viral DNA and mRNA in the cell population. However, the average viral DNA and mRNA content in a heterogeneous cell population does not inevitably reflect the abundance in individual cells. As the vast majority of virus infection studies is carried out using standard experimental procedures with heterogeneous cell populations, there is a need for a method allowing simultaneous detection and quantitative analysis of viral genome accumulation and gene expression in individual infected cells within a population. This article describes a padlock probe-based rolling-circle amplification protocol that allows simultaneous detection of HAdV type 5 (HAdV-5) DNA and various virus-encoded mRNAs, as well as quantitative analysis of HAdV-5 DNA copies and mRNA species, in individual cells within a heterogeneous population. This versatile method can be used to detect the extent of pathogenic DNA virus infection in different cell types over prolonged infection times. Furthermore, simultaneous viral DNA and mRNA quantification in individual cells allows identification of cells in which persistent infections may be established. © 2018 by John Wiley & Sons, Inc., (Copyright © 2018 John Wiley & Sons, Inc.)

Quantitative DNA amplification using fluorescence labeling has played an important role in the recent, rapid progress of basic medical and molecular biological research. Here we report a label-free detection of real-time DNA amplification using a nanofluidic diffraction grating. Our detection system observed intensity changes during DNA amplification of diffracted light derived from the passage of a laser beam through nanochannels embedded in a microchannel. Numerical simulations revealed that the diffracted light intensity change in the nanofluidic diffraction grating was attributed to the change of refractive index. We showed the first case reported to date for label-free detection of real-time DNA amplification, such as specific DNA sequences from tubercle bacilli (TB) and human papillomavirus (HPV). Since our developed system allows quantification of the initial concentration of amplified DNA molecules ranging from 1 fM to 1 pM, we expect that it will offer a new strategy for developing fundamental techniques of medical applications.

Rolling circle amplification (RCA) for generation of distinct fluorescent signals in situ relies upon the self-collapsing properties of single-stranded DNA in commonly used RCA-based methods. By introducing a cross-hybridizing DNA oligonucleotide during rolling circle amplification, we demonstrate that the fluorophore-labeled RCA products (RCPs) become smaller. The reduced size of RCPs increases the local concentration of fluorophores and as a result, the signal intensity increases together with the signal-to-noise ratio. Furthermore, we have found that RCPs sometimes tend to disintegrate and may be recorded as several RCPs, a trait that is prevented with our cross-hybridizing DNA oligonucleotide. These effects generated by compaction of RCPs improve accuracy of visual as well as automated in situ analysis for RCA based methods, such as proximity ligation assays (PLA) and padlock probes.

We demonstrate detection of DNA coils formed from a Vibrio cholerae DNA target at picomolar concentrations using a novel optomagnetic approach exploiting the dynamic behavior and optical anisotropy of magnetic nanobead (MNB) assemblies. We establish that the complex second harmonic optical transmission spectra of MNB suspensions measured upon application of a weak uniaxial AC magnetic field correlate well with the rotation dynamics of the individual MNBs. Adding a target analyte to the solution leads to the formation of permanent MNB clusters, namely, to the suppression of the dynamic MNB behavior. We prove that the optical transmission spectra are highly sensitive to the formation of permanent MNB clusters and, thereby to the target analyte concentration. As a specific clinically relevant diagnostic case, we detect DNA coils formed via padlock probe recognition and isothermal rolling circle amplification and benchmark against a commercial equipment. The results demonstrate the fast optomagnetic readout of rolling circle products from bacterial DNA utilizing the dynamic properties of MNBs in a miniaturized and low-cost platform requiring only a transparent window in the chip.

Rotavirus infections are one of the most common reasons for hospitalizations due to gastrointestinal diseases. Rotavirus is often diagnosed by latex agglutination assay, chromatography immunoassay, or by electron microscopy, which are all quite insensitive. Reverse transcription polymerase chain reaction, on the other hand, is very sensitive to variations at the genomic level. We developed a novel assay based on a set of 58 different padlock probes with a detection limit of 1,000 copies. Twenty-two patient samples were analyzed and the assay showed high concordance with a PCR-based assay. In summary, we present a new assay for sensitive and variation tolerant detection of rotavirus.

We demonstrate a novel digital microfluidic nucleic acid amplification concept which is based on padlock probe mediated DNA detection and isothermal circle-to-circle amplification (C2CA). This assay platform combines two digital approaches. First, digital microfluidic manipulation of droplets which serve as micro-reaction chambers and shuttling magnetic particles between these droplets facilitates the integration of complex solid phase multistep assays. We demonstrate an optimized novel particle extraction and transfer protocol for superparamagnetic particles on a digital microfluidic chip that allows for nearly 100% extraction efficiencies securing high assay performance. Second, the compartmentalization required for digital single molecule detection is solved by simple molecular biological means, circumventing the need for complex microfabrication procedures necessary for most, if not all, other digital nucleic acid detection methods. For that purpose, padlock probes are circularized in a strictly target dependent ligation reaction and amplified through two rounds of rolling circle amplification, including an intermediate digestion step. The reaction results in hundreds of 500 nm sized individually countable DNA nanospheres per detected target molecule. We demonstrate that integrated miniaturized digital microfluidic C2CA results in equally high numbers of C2CA products μL(-1) as off-chip tube control experiments indicating high assay performance without signal loss. As low as 1 aM synthetic Pseudomonas aeruginosa DNA was detected with a linear dynamic range over 4 orders of magnitude up to 10 fM proving excellent suitability for infectious disease diagnostics.

In situ padlock/rolling circle amplification (RCA) is a method used to amplify, visualize, and quantify target DNA molecules in cells. However, the multiple reaction steps involved make this technique costly and cumbersome. We developed a novel, simplified, automated microfluidic system for RCA, and demonstrated its effectiveness by counting amplified mitochondrial DNA fragments in HeLa cells. After optimizing the volume of the reaction solutions and washing buffer composition, the product yield was equal to that obtained by the conventional manual method. The required volume of reagents was reduced to 10 μL, which is less than half the volume used in the conventional method. To the best of our knowledge, this is the first report of an automated microfluidic method for in situ padlock/RCA, which can be useful for making highly efficient pathological diagnoses.

Padlock rolling circle amplification (RCA) is a powerful analytical method for ultrasensitive DNA detection. Although there are some advantages to bead-based RCA, a detailed study of the relationship between the bead material and the efficiency of bead-based RCA has not been reported. Here, we compared the reaction efficiencies of bead-based RCA performed on two types of bead material: agarose and polystyrene. Agarose was a more suitable material for on-bead RCA. The calibration curve showed linearity between 0.05 and 1 nM, and the limit of detection was 9 pM (9 amol) for Salmonella DNA determination., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Crimean Congo hemorrhagic fever virus (CCHFV) is a human pathogen that causes a severe disease with high fatality rate for which there is currently no specific treatment. Knowledge regarding its replication cycle is also highly limited. In this study we developed an in situ technique for studying the different stages during the replication of CCHFV. By integrating reverse transcription, padlock probes, and rolling circle amplification, we were able to detect and differentiate between viral RNA (vRNA) and complementary RNA (cRNA) molecules, and to detect viral protein within the same cell. These data demonstrate that CCHFV nucleocapsid protein (NP) is detectable already at 6 hours post infection in vRNA- and cRNA-positive cells. Confocal microscopy showed that cRNA is enriched and co-localized to a large extent with NP in the perinuclear area, while vRNA has a more random distribution in the cytoplasm with only some co-localize with NP. However, vRNA and cRNA did not appear to co-localize directly., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Detection and identification of pathogens in environmental samples for biosecurity applications are challenging due to the strict requirements on specificity, sensitivity and time. We have developed a concept for quick, specific and sensitive pathogen identification in environmental samples. Target identification is realized by padlock- and proximity probing, and reacted probes are amplified by RCA (rolling-circle amplification). The individual RCA products are labeled by fluorescence and enumerated by an instrument, developed for sensitive and rapid digital analysis. The concept is demonstrated by identification of simili biowarfare agents for bacteria (Escherichia coli and Pantoea agglomerans) and spores (Bacillus atrophaeus) released in field.

We developed a molecular diagnostic method for detection of RNA virus based on padlock probes and colorimetric readout. The feasibility of our approach was demonstrated by using detection of Crimean-Congo hemorrhagic fever (CCHF) virus as a model. Compared with conventional PCR-based methods, our approach does not require advanced equipment, involves easier assay design, and has a sensitivity of 10(3) viral copies/ml. By using a cocktail of padlock probes, synthetic templates representing different viral strain variants could be detected. We analyzed 34 CCHF patient samples, and all patients were correctly diagnosed when the results were compared to those of the current real-time PCR method. This is the first time that highly specific padlock probes have been applied to detection of a highly variable target sequence typical of RNA viruses.

The rolling circle amplification (RCA) is a versatile DNA amplification method in which a DNA molecule is amplified using a single DNA primer, allowing the product to be counted as a single dot. Circular templates for RCA can arise from padlock probes in highly specific DNA target-mediated ligation reactions. However, improvement of detection efficiency represents an important challenge. In homogeneous assays, the detection efficiency is generally only under 0.1%, mainly because the sample volume is too large compared with the detection volume. Here, we used microchannel surfaces in a glass microchip for DNA detection in small volume samples. First, DNA patterning on glass surfaces in microchannels was demonstrated using chemical surface patterning by UV light. By using a photochemical reaction, we realized DNA patterning in a closed space. Second, RCA was demonstrated using dilutions of target molecules, and a calibration curve was obtained. The highest detection efficiency was 22.5% by virtue of the reduced sample volumes from several hundred microliters to 5.0 nL. Accordingly, a countable number of DNA molecules was successfully detected. This method is suitable for analysis of very small volume samples such as single cells, especially by using extended-nanochannels with dimensions of 10-1000 nm.

Background: In this study we utilized padlock probes and rolling circle amplification as a mean to detect and study the replication of porcine circovirus type 2 (PCV2) in cultured cells and in infected tissue. Porcine circovirus type 2 is a single-stranded circular DNA virus associated with several severe diseases, porcine circovirus diseases (PCVD) in pigs, such as postweaning multisystemic wasting syndrome. The exact reason and mechanisms behind the trigger of PCV2 replication that is associated with these diseases is not well-known. The virus replicates with rolling circle replication and thus also exists as a double-stranded replicative form., Results: By applying padlock probes and rolling circle amplification we could not only visualise the viral genome but also discriminate between the genomic and the replicative strand in situ. The genomic strand existed in higher numbers than the replicative strand. The virus accumulated in certain nuclei but also spread into the cytoplasm of cells in the surrounding tissue. In cultured cells the average number of signals increased with time after infection., Conclusions: We have developed a method for detection of both strands of PCV2 in situ that can be useful for studies of replication and in situ detection of PCV2 as well as of DNA viruses in general.

The sensitive detection and quantification of DNA targets in the food industry and in environmental and clinical settings are issues of utmost importance in ensuring contamination-free food, monitoring the environment, and battling disease. Selective probes coupled with powerful amplification techniques are therefore of major interest. In this study, we set out to create an integrated microchemical chip that benefits from microfluidic chip technology in terms of sensitivity and a strong detection methodology provided jointly by padlock probes and rolling circle amplification (RCA). Here, we have integrated padlock probes and RCA into a microchip. The chip uses solid phase capture in a microchannel to enable washing cycles and decrease analytical area, and employs on-bead RCA for single-molecule amplification and detection. We investigated the effects of reagent concentration and amount of padlock probes, and demonstrated the feasibility of detecting Salmonella.

Increasing knowledge about the heterogeneity of mRNA expression within cell populations highlights the need to study transcripts at the level of single cells. We present a method for detection and genotyping of individual transcripts based on padlock probes and in situ target-primed rolling-circle amplification. We detect a somatic point mutation, differentiate between members of a gene family and perform multiplex detection of transcripts in human and mouse cells and tissue.

A subtyping assay for both the hemagglutinin (HA) and neuraminidase (NA) surface antigens of the avian influenza virus (AIV) has been developed. The method uses padlock probe chemistry combined with a microarray output for detection. The outstanding feature of this assay is its capability to designate both the HA and the NA of an AIV sample from a single reaction mixture. A panel of 77 influenza virus strains was tested representing the entire assortment of the two antigens. One hundred percent (77/77) of the samples tested were identified as AIV, and 97% (75/77) were subtyped correctly in accordance with previous examinations performed by classical diagnostic methods. Testing of heterologous pathogens verified the specificity of the assay. This assay is a convenient and practical tool for the study of AIVs, providing important HA and NA data more rapidly than conventional methods.

We have developed a new method for a fast and precise analysis of circle-to-circle amplification (C2CA) product for specific gene detection by microchip electrophoresis. In this method, we have added a new enzymatic step to the C2CA reaction, which could be carried out isothermally at 37 degrees C. Compared to the original single-stranded DNA, the double-stranded DNA that is produced by this enzymatic reaction is more reliable for analysis by microchip electrophoresis. C2CA product was detected within 55 s with high reproducibility by this method which was successfully applied to the detection of 10-ng genomic DNA of the pathogenic bacteria Vibrio. cholerae within 110 s. Purification was found to be an indispensable step for the analysis of the C2CA product of genomic DNA samples.

We have developed a procedure for massively parallel resequencing of multiple human genes by combining a highly multiplexed and target-specific amplification process with a high-throughput parallel sequencing technology. The amplification process is based on oligonucleotide constructs, called selectors, that guide the circularization of specific DNA target regions. Subsequently, the circularized target sequences are amplified in multiplex and analyzed by using a highly parallel sequencing-by-synthesis technology. As a proof-of-concept study, we demonstrate parallel resequencing of 10 cancer genes covering 177 exons with average sequence coverage per sample of 93%. Seven cancer cell lines and one normal genomic DNA sample were studied with multiple mutations and polymorphisms identified among the 10 genes. Mutations and polymorphisms in the TP53 gene were confirmed by traditional sequencing.

In this review I will describe the development of a technique that enables genotyping of individual DNA molecules in the context of morphologically preserved fixed cells, from the fundamental concept published in 1994 to the present status. The review describes enzyme-assisted histochemistry approaches to achieve highly specific molecular identification reactions coupled to efficient signal amplification. The primary molecular identification is accomplished through circularization of oligonucleotide probes, called padlock probes. The circularization reaction is catalyzed by a DNA ligase, which provides robust distinction between single-nucleotide variants under standard reaction conditions. To generate a detectable signal from individual circularized probe molecules, a DNA polymerase is added that replicates probe circles, generating a long tandem-repeated DNA product, easily visualized using a standard epi-fluorescence microscope. Individual signals are recorded as bright dots, providing digital information about the abundance of specific sequences and opportunities for simultaneous detection of several targets using spectral multiplexing. The importance of strictly target-dependent signal amplification will be discussed.

During the past two years, significant breakthroughs have been achieved in genetic analyses through the application of technologies based on analytical DNA-circularization reactions. Padlock probes and molecular inversion probes have enabled parallel, high-throughput single nucleotide polymorphism (SNP) genotyping at increased scales, whereas, at the other end of the analysis spectrum, DNA molecules in individual cells have been genotyped, in situ, using padlock probes and rolling-circle amplification (RCA). This review describes the recent developments in the technologies that use specific DNA circularization, coupled to DNA amplification through PCR or rolling-circle amplification, and addresses the great potential of these tools.

Methods are needed to study single molecules to reveal variability, interactions and mechanisms that may go undetected at the level of populations of molecules. We describe here an integrated series of reaction steps that allow individual nucleic acid molecules to be detected with excellent specificity. Oligonucleotide probes are circularized after hybridization to target sequences that have been prepared so that localized amplification reactions can be initiated from the target molecules. The process results in strong, discrete detection signals anchored to the target molecules. We use the method to observe the distribution, within and among human cells, of individual normal and mutant mitochondrial genomes that differ at a single nucleotide position.

We describe a method to monitor rolling-circle replication of circular oligonucleotides in dual-color and in real-time using molecular beacons. The method can be used to study the kinetics of the polymerization reaction and to amplify and quantify circularized oligonucleotide probes in a rolling-circle amplification (RCA) reaction. Modified molecular beacons were made of 2'-O-Me-RNA to prevent 3' exonucleolytic degradation by the polymerase used. Moreover, the complement of one of the stem sequences of the molecular beacon was included in the RCA products to avoid fluorescence quenching due to inter-molecular hybridization of neighboring molecular beacons hybridizing to the concatemeric polymerization product. The method allows highly accurate quantification of circularized DNA over a broad concentration range by relating the signal from the test DNA circle to an internal reference DNA circle reporting in a distinct fluorescence color.