Rolling Circle Amplification Assay Research Articles

Multispectral pathogens detection in food using multiplex hyperbranched saltatory rolling circle amplification

Foodborne illnesses caused by Salmonella and Staphylococcus aureus are a significant public health concern, leading to societal and economic repercussions. It is important to develop a simple and straightforward bacteria detection and identification method. A triple-probe multiplex rolling circle amplification technique has been developed to simultaneously detect Salmonella Typhimurium and S. aureus. This method utilizes fluorophore-labeled long padlock probes targeting S. Typhimurium invA and S. aureus glnA specific genes, along with a pH-based detection approach for direct visual identification. The multiplex hyperbranched saltatory rolling circle amplification assay at 30 °C has showed promising results with synthetic targets within 30 min and real bacteria within 2 h after establishing the detection settings. The assay is specific for S. aureus and S. Typhimurium, with a limit of detection of 39 μM for fluorescence and 78 μM for colorimetric. In the simulative test of this method for the detection of S. Typhimurium and S. aureus in milk, the limit of detection for the fluorescence signal after 2 h of amplification was 10 CFU/mL and 5 CFU/mL, respectively. The detection method was evaluated to be stable enough to detect pathogen for 3.29 months. Consequently, this triple-probe-multiplex rolling circle amplification method displays notable specificity, sensitivity, as well as ease of interpretation when testing food samples for harmful pathogens.

Closed-tube saltatory rolling circle amplification assay for rapid and visual detection of allergenic cashew nut in foods

Closed-tube saltatory rolling circle amplification assay for rapid and visual detection of allergenic cashew nut in foods

DSN enzyme recognition initiated rolling circle amplification (DiRCA) for accurate miRNA detection in immune response and immune repair after trauma

Recent studies have reported that miRNA plays an important role in immune response and immune repair after trauma. By regulating the expression of related target genes, miRNA regulates the production, proliferation, development and immune response of immune cells. Therefore, it is in urgent demand to develop an novel method for miRNA analysis. Rolling circle amplification (RCA), as an attractive isothermal signal amplification strategy, has been widely utilized in constructing miRNA detection assays. However, accurate and sensitive miRNA quantitative determination remains a huge challenge for RCA based approaches. Herein, we propose a DSN enzyme based signal cycle initiated Rolling Circle Amplification assay (DiRCA) for sensitive and accurate miRNA detection. In DiRCA, target miRNA unfolds hairpin structure probe in the detection scaffold, forming a RNA–DNA duplex. DSN enzyme is utilized to specifically digest the DNA sequence in RNA–DNA duplex, releasing miRNA to form a signal cycle; its capability to distinguish one base pair mismatch in RNA–DNA duplex endows DiRCA a high selectivity. Meanwhile, DSN enzyme based cleavage initiates RCA, transcribing G-rich sequences for signal generation. Based on the DSN assisted signal cycle and RCA, DiRCA shows a low limit of detection of 0.43 fM and a superior capability in selectively detecting mismatched miRNA sequences, showing a promising prospect in the early-diagnosis of disease.

Long-Term Culture of Canine Ocular Cells That Maintain Canine Papillomaviruses.

Canine ocular papillomas occur on the haired skin of eyelids, conjunctival epithelium, and rarely on the cornea. Using PCR typing assays with canine papillomavirus type-specific primer sets, our study confirmed that the papillomas contained canine papillomavirus type 1. The positive result from a rolling circle amplification assay indicated the CPV1 viral genome in the cells is a circular episomal form. We also successfully established the first canine corneal cell line using the conditional reprogramming method. The cells exhibited an epithelial cell morphology, grew rapidly in vitro, and could be maintained long term. For the continued growth of the canine corneal cells, feeder cells played a more important role than Rho-kinase inhibitor Y-27632. More importantly, the viral CPV1 genome was maintained in the canine corneal cells during the long-term expansion. Unlimited supplies of these cells provide as a model for the study CPV in dog cells, and a platform for drug screening for effective therapies against canine papillomavirus infection in the future.

Saltatory rolling circle amplification assay for simple and visual detection of Listeria monocytogenes in milk and milk products

A novel saltatory rolling circle amplification (SRCA) assay was established for prompt identification of Listeria monocytogenes in milk and Indian milk cake (kalakhand). The SRCA and conventional PCR were independently able to detect 63 fg and 630 fg L. monocytogenes DNA per reaction, respectively. The detection limit of SRCA was evaluated in artificially contaminated milk and kalakhand. In milk, without any enrichment, SRCA was able to detect 4.4 × 102 cfu mL−1 of milk, indicating 100-fold greater sensitivity than PCR (4.4 × 104 cfu mL−1). After enrichment up to 3 h and 6 h, detection in SRCA improved to 4.4 cfu mL−1 of milk. Spiking studies in kalakhand showed 10-times better results for SRCA than PCR. The study illustrates that the developed SRCA-based test is sensitive, cost-effective, rapid, and most importantly simple for the detection of L. monocytogenes and it can be adopted for on-site detection of L. monocytogenes in a variety of milk and milk products.

Blocker-tailed PCR coupled with rolling circle amplification for fluorescent detection of emetic Bacillus cereus in milk

Emetic Bacillus cereus (B. cereus) is a foodborne microorganism associated with vomiting disease. Herein, we have established a blocker-tailed polymerase chain reaction (bt-PCR) coupled with rolling circle amplification (RCA) assay for the sensitive and specific detection of emetic B. cereus. In this research, the designed forward primer for bt-PCR was modified into two parts, namely, the C3 spacer blocker, and the linker DNA binding sequence, which was complementary with the linker DNA. The bt-PCR products could bind to the linker DNA and initiate the RCA assay, generating numerous repetitive G-quadruplex structures. The specific fluorescence dye of thioflavin T (ThT) could bind to the G-quadruplex structures, producing enhanced fluorescent signals for detection. Under optimized conditions, the proposed assay could improve the sensitivity of the conventional PCR detection, and the limit of detection (LOD) was 2.6 × 100 CFU/mL in pure culture, and 2.8 × 100 CFU/mL in spiked milk samples with good specificity. The proposed assay showed potential application for the detection of emetic B. cereus in real world.

Rolling Circle Amplification-Coupled Glass Nanopore Counting of Mild Traumatic Brain Injury-Related Salivary miRNAs.

Mild traumatic brain injury (mTBI) could be underdiagnosed and underreported due to the delayed onset of symptoms and the conventional subjective assessment. Recent studies have suggested that salivary microRNAs (miRNAs) could be reliable biomarkers for objective mTBI diagnosis. In this work, we demonstrated a rolling circle amplification (RCA)-coupled resistive pulse-counting platform for profiling mTBI-related miRNAs, using easy-to-fabricate large glass nanopores (200 nm diameter). The method relies on the linear and specific elongation of the miRNA to a much larger RCA product, which the large glass nanopore can digitally count with a high signal-to-noise ratio. We developed and validated the RCA assay against let-7a, miR-30e, and miR-21. We demonstrated the quantification capability of this large glass nanopore counting platform for purified miRNAs as well as miRNAs in salivary total RNA background. Finally, we quantitatively evaluated the performance of profiling each individual miRNAs in a mixed analyte. Our results showed that the RCA-coupled large glass nanopore counting provides a promising and accessible alternative toward the clinical diagnosis of mTBI using salivary miRNAs.

Rolling Circle Amplification as an Efficient Analytical Tool for Rapid Detection of Contaminants in Aqueous Environments.

Environmental contaminants are a global concern, and an effective strategy for remediation is to develop a rapid, on-site, and affordable monitoring method. However, this remains challenging, especially with regard to the detection of various contaminants in complex water environments. The application of molecular methods has recently attracted increasing attention; for example, rolling circle amplification (RCA) is an isothermal enzymatic process in which a short nucleic acid primer is amplified to form a long single-stranded nucleic acid using a circular template and special nucleic acid polymerases. Furthermore, this approach can be further engineered into a device for point-of-need monitoring of environmental pollutants. In this paper, we describe the fundamental principles of RCA and the advantages and disadvantages of RCA assays. Then, we discuss the recently developed RCA-based tools for environmental analysis to determine various targets, including heavy metals, organic small molecules, nucleic acids, peptides, proteins, and even microorganisms in aqueous environments. Finally, we summarize the challenges and outline strategies for the advancement of this technique for application in contaminant monitoring.

A simple and sensitive aptasensor with rolling circle amplification for viable Cronobacter sakazakii detection in powdered infant formula

Cronobacter sakazakii is a foodborne, emerging opportunistic pathogen that causes severe bacteremia, necrotizing enterocolitis, and sepsis with a mortality rate of up to 80%. In this study, we developed a simple and sensitive fluorescent turn-off aptasensor with rolling circle amplification assay for viable C. sakazakii detection in powdered infant formula. The results showed that the proposed aptasensor has good performance and specificity for detecting viable C. sakazakii in pure culture and powdered infant formula samples within 3 h. Under the optimal reaction conditions, there is a linear relationship between fluorescent intensity at 490 nm and logarithmic concentration of C. sakazakii in the range of 2.7 × 105 to 2.7 × 102 cfu/mL, with a limit of detection of 2.7 × 102 cfu/mL in pure culture. The proposed aptasensor achieved a recovery of 104 to 111% in pure culture, and 96 to 107% in spiked powdered infant formula samples. The proposed aptasensor does not require complicated DNA extraction steps or antibodies, and can be performed at 37°C, making it a convenient and sensitive strategy for C. sakazakii detection.

A logic dual-channel detection of Hox transcript antisense intergenic RNA using graphene switch and padlock probe-based exponential rolling circle amplification assay

Sensitive and simultaneous detection of multiple low-abundance long noncoding RNA (lncRNA) biomarkers promises an early diagnosis of lung cancer. In this work, we demonstrate a quadruple logic-mode (YES, NOT, AND and OR) sensing platform for dual specific sequences (T1 and T2) of Hox transcript antisense intergenic RNA (HOTAIR) based on padlock probe-based exponential rolling circle amplification (P-ERCA) assay, producing dual-channel electrochemical and fluorescence (FL) signals simultaneously (AND/OR mode). T1/T2 acts as a key unlocking the P-ERCA amplification strategy, combining with fluorescein Cy3/Cy5 and Pb2+/Cd2+ labeled polydopamine nanospheres (PDA-Pb2+/PDA-Cd2+) give out the dual-channel signals, realizing the quantitative detection of T1 and T2. The dual-channel assay exhibits a wide linear range of 1 fM to 100 pM with low detection limits of 0.25 fM and 0.3 fM for these two targets, respectively. Furthermore, the proposed genosensor possesses high sensitivity, selectivity, and anti-interference ability. With standard addition method, our work shows the recovery experimental T1 value of 86–114.6 % and T2 of 82.2–159 % in diluted serum samples. Moreover, the proposed genosensor obtained comparable detection results of HOTAIR content in whole RNA extracted from the real blood samples with those performed by standard qRT-PCR technique. Results in this study confirmed the feasibility of using the HOTAIR genosensing strategy in bioanalysis.

Development of novel visual detection methodology for Salmonella in meat using saltatory rolling circle amplification.

The aim of this study was to develop a saltatory rolling circle amplification (SRCA) assay for rapid, simple and visual detection of Salmonella in meat. Saltatory rolling circle amplification assay was established using simple PCR primers targeting the invA gene of Salmonella enterica. The specificity of the SRCA assay was determined using 28 Salmonella and 15 non-Salmonella strains. The analytical sensitivity of the developed SRCA, conventional and real-time PCR assays were 70fg, 7pg and 700fg S. enterica DNA per tube, respectively. The limit of detection (LoD) of the SRCA assay was 40CFU per gram of meat without enrichment and 4CFU per gram after including 6h brief enrichment step. The detection limits of 40CFU per gram and 4CFU per gram of meat were achieved within 165min and 9h, respectively (including DNA extraction). To assess the real-world relevance of the SRCA assay, it was used to screen Salmonella from the field pork samples (n=82). The same samples were also tested with culture (ISO 6579: 2002) method, conventional and real-time PCR assays. Using the developed assay with 6-h enrichment step, it could give accurate results as that of the culture method. The results of this study showed that the SRCA assay is a rapid, simple, sophisticated equipment-free and user-friendly method for accurate detection of Salmonella in meat foods. To our information, this is the first study to deploy SRCA assay for screening foods for Salmonella. The developed SRCA assay is cost-effective, easy-to-perform and equipment-free; therefore, it has the potential to replace other molecular detection methods for regular screening of Salmonella in foods in field laboratories.

Quantification and epigenetic evaluation of the residual pool of hepatitis B covalently closed circular DNA in long-term nucleoside analogue-treated patients

Hepatitis B virus (HBV) covalently closed circular (ccc)DNA is the key genomic form responsible for viral persistence and virological relapse after treatment withdrawal. The assessment of residual intrahepatic cccDNA levels and activity after long-term nucleos(t)ide analogues therapy still represents a technical challenge. Quantitative (q)PCR, rolling circle amplification (RCA) and droplet digital (dd)PCR assays were used to quantify residual intrahepatic cccDNA in liver biopsies from 56 chronically HBV infected patients after 3 to 5 years of telbivudine treatment. Activity of residual cccDNA was evaluated by quantifying 3.5 kB HBV RNA (preC/pgRNA) and by assessing cccDNA-associated histone tails post-transcriptional modifications (PTMs) by micro-chromatin immunoprecipitation. Long-term telbivudine treatment resulted in serum HBV DNA suppression, with most of the patients reaching undetectable levels. Despite 38 out of 56 patients had undetectable cccDNA when assessed by qPCR, RCA and ddPCR assays detected cccDNA in all-but-one negative samples. Low preC/pgRNA level in telbivudine-treated samples was associated with enrichment for cccDNA histone PTMs related to repressed transcription. No difference in cccDNA levels was found according to serum viral markers evolution. This panel of cccDNA evaluation techniques should provide an added value for the new proof-of-concept clinical trials aiming at a functional cure of chronic hepatitis B.

Protein-Mediated Suppression of Rolling Circle Amplification for Biosensing with an Aptamer-Containing DNA Primer.

We report a method to detect proteins via suppression of rolling circle amplification (RCA) by using an appropriate aptamer as the linear primer (denoted as an aptaprimer) to initiate RCA. In the absence of a protein target, the aptaprimer is free to initiate RCA, which can produce long DNA products that are detected via binding of a fluorescent intercalating dye. Introduction of a target causes the primer region within the aptamer to become unavailable for binding to the circular template, inhibiting RCA. Using SYBR Gold or QuantiFluor dyes as fluorescent probes to bind to the RCA reaction product, it is possible to produce a generic protein-modulated RCA assay system that does not require fluorophore- or biotin-modified DNA species, substantially reducing complexity and cost of reagents. Based on this modulation of RCA, we demonstrate the ability to produce both solution and paper-based assays for rapid and quantitative detection of proteins including platelet derived growth factor and thrombin.

Automated on-chip analysis of tuberculosis drug-resistance mutation with integrated DNA ligation and amplification.

Detection of a single base mutation in Mycobacterium tuberculosis DNA can provide fast and highly specific diagnosis of antibiotic-resistant tuberculosis. Mutation-specific ligation of padlock probes (PLPs) on the target followed by rolling circle amplification (RCA) is highly specific, but challenging to integrate in a simple microfluidic device due to the low temperature stability of the phi29 polymerase and the interference of phi29 with the PLP annealing and ligation. Here, we utilized the higher operation temperature and temperature stability of Equiphi29 polymerase to simplify the integration of the PLP ligation and RCA steps of an RCA assay in two different strategies performed at uniform temperature. In strategy I, PLP annealing took place off-chip and the PLP ligation and RCA were performed in one pot and the two reactions were clocked by a change of the temperature. For a total assay time of about 1.5h, we obtained a limit of detection of 2pM. In strategy II, the DNA ligation mixture and the RCA mixture were separated into two chambers on a microfluidic disc. After on-disc PLP annealing and ligation, the disc was spun to mix reagents and initiate RCA. For a total assay time of about 2h, we obtained a limit of detection of 5pM. Graphical abstract.

Dual-signal amplification strategy: Universal asymmetric tailing-PCR triggered rolling circle amplification assay for fluorescent detection of Cronobacter spp. in milk

Cronobacter spp. are important opportunistic foodborne pathogens in powdered infant formula that cause many serious diseases in neonates and infants. In this study, a novel assay based on dual signal amplification strategy was developed by coupling asymmetric tailing PCR (AT-PCR) with rolling circle amplification (RCA) for the detection of Cronobacter spp. in milk. The tailing single-stranded DNA was generated through AT-PCR and used to initiate RCA, generating tandem repetitive G-quadruplex sequences. In the presence of the fluorescence dye thioflavin T that could intercalate into the G-quadruplex structures, the fluorescence signal was detected with a microplate reader. The AT-PCR coupled with RCA assay was specific for Cronobacter spp. detection because of the highly specific primers chosen for the AT-PCR. The limits of detection were 4.3 × 101 cfu/mL in pure culture and 4.5 × 102 cfu/mL in spiked milk, respectively. The fixed sequences designed in the hairpin DNA allowed this AT-PCR coupled with RCA assay to serve as a universal platform for the detection of other pathogens by modifying the specificity of the PCR primers.

Digital Rolling Circle Amplification–Based Detection of Ebola and Other Tropical Viruses

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.

Optomagnetic Detection of Rolling Circle Amplification Products.

Rolling circle amplification (RCA) of a synthetic nucleic acid target is detected using magnetic nanoparticles (MNPs) combined with an optomagnetic (OM) readout. Two RCA assays are developed with on-chip detection of rolling circle products (RCPs) either at end-point where MNPs are mixed with the sample after completion of RCA or in real time where MNPs are mixed with the sample during RCA. The plastic chip acts as a cuvette, which is positioned in a setup integrated with temperature control and simultaneous detection of four parallel DNA hybridization reactions between functionalized MNPs and products of DNA amplification. The OM technique probes the small-angle rotation of MNPs bearing oligonucleotide probes complementary to the repeated nucleotide sequence of the RCPs. This rotation is restricted when MNPs bind to RCPs, which can be observed as a turn-off of the signal from MNPs that are free to rotate. The amount of MNPs bound to RCPs is found to increase in response to the amplification time as well as in response to the synthetic DNA target concentration (2-40pM dynamic range). We report OM real-time results obtained with MNPs present during RCA and compare to relevant end-point OM results for RCPs generated for different RCA times. The real-time approach avoids opening of tubes post-RCA and thus reduces risk of lab contamination with amplification products without compromising the sensitivity and dynamic range of the assay.

Integration of microbead DNA handling with optomagnetic detection in rolling circle amplification assays.

Rolling circle amplification (RCA) is a linear isothermal amplification technique that is widely applied in biomolecular assays due to its high specificity. Handling of a target sample using magnetic microbeads (MMBs) in a multi-step assay is appealing as the MMBs enable separation and transportation using an external magnet. Detection of amplicons using optomagnetic measurements of the rotational diffusion properties of magnetic nanoparticles (MNPs) is also appealing as it can be performed on any transparent sample container. Two strategies are described for integration of MMB sample handling in an RCA assay with on-chip optomagnetic detection of the amplification products. The first strategy relies on selective and irreversible release of the amplicons from the MMBs so that the binding of functionalized MNPs to the amplicons can be detected optomagnetically. The second strategy relies on the incorporation of MNPs into RCA products during RCA, followed by their separation on MMBs and subsequent optomagnetic detection upon release from the RCA products. Using MMB handling of RCA steps, the limits of detection (LODs) for a synthetic DNA target representative of Victoria Influenza type B were found to be between 4 and 20 pM with total assay times between 2 and 2.5 h. Without magnetic microbead sample handling, the LOD was 200 fM. The findings provide deeper insight into the use of magnetic microbeads as solid substrates to handle a DNA target for integration of RCA as well as other DNA-based assays. Graphical Abstract Schematic illustration of magnetic microbeads transporting a DNA target through the steps in a rolling circle amplification assay. Optomagnetic measurements detect the binding of magnetic nanoparticles to amplicons released from microbeads (top) or the pH-induced release of magnetic nanoparticles trapped in amplicons (bottom).

Integration of rolling circle amplification and optomagnetic detection on a polymer chip

Rolling circle amplification (RCA) combined with padlock probe recognition of a DNA target is attractive for on-chip nucleic acid testing due to its high specificity and isothermal reaction conditions. However, the integration of RCA on an automated chip platform is challenging due to the different reagents needed for the reaction steps and the temperature sensitivity of the phi29 polymerase. Here, we describe the integration of an RCA assay on a single-use polymer chip platform where magnetic microbeads are used as solid support to transport the DNA target between three connected reaction chambers for (i) padlock probe annealing and ligation, (ii) RCA, and (iii) optomagnetic detection of RCA products. The three chambers were loaded with reagents by sequential filling combined with passive microfluidic structures. After loading, the on-chip assay steps were automated. For an assay in which all steps but the padlock probe annealing on the target were performed on-chip, we found a limit of detection (LOD) for a synthetic influenza target of 2 pM after 45 min of RCA, which is comparable to the corresponding laboratory assay. The entire assay, including padlock probe annealing, could be performed on-chip with an LOD of 20 pM after 45 min of RCA. This LOD can likely be reduced by further optimizing the microbead mixing. The results present important steps towards the integration and automation of RCA and potentially also other complex multi-step assays on a single-use polymer chip for molecular analysis.

SpliceRCA: in Situ Single-Cell Analysis of mRNA Splicing Variants

Immune cell heterogeneity due to the differential expression of RNA splicing variants still remains unexplored. This is mainly because single-cell imaging technologies of splicing variants with precise sequence or base resolution are now not readily available. Herein, we design a splice-junction anchored padlock-probe-mediated rolling circle amplification assay (SpliceRCA) for single-cell imaging of splice isoforms of essential regulatory immune gene (CD45) upon T-cell activation. Two recognition regions in the padlock probe can target the splice-junction sequence, resulting in a close proximity for triggering in situ one-target-one-amplicon amplification. With the read length of ∼30 nucleotides, this method allows discrimination of isoforms with single-base precision and quantification of isoforms with single-molecule resolution. We applied SpliceRCA to single-cell image splice variants of essential regulatory immune gene (CD45) upon T-cell activation. It is found that CD45RO isoform presents a distal nuclear spatial distribution and is coregulated with CD45RB upon activation. Our strategy provides a single-cell analysis platform to investigate the mechanism of complex immune responses and may further guide immunotherapy.