Rolling Circle Amplification Products Research Articles

Rapid and ultrasensitive miRNA detection by combining endonuclease reactions in a rolling circle amplification (RCA)-based hairpin DNA fluorescent assay.

MicroRNA (miRNA) sensing strategies employing rolling circle amplification (RCA) coupled with the hairpin DNA (HD) probe-mediated FRET assay have shown promise, but achieving rapid, sensitive, and specific detection of target miRNA remains a challenge in clinical diagnostics. Herein, we incorporate PstI endonuclease cleavage (PEC) into a conventional RCA-based HD probe FRET assay to develop an effective and feasible method. Long single-stranded RCA products are synthesized from miRNA-21 loaded on a circular dumbbell DNA, and the resultant RCA products self-assemble to generate long HD structures with double-stranded stem regions that are specifically recognized and cleaved by PstI endonucleases when incubated with PstI enzymes. This releases large amounts of short single-stranded DNA fragments that hybridize and open to the complementary loop-stem regions of HD probes labeled with FAM at one end and BHQ-1 at the other, resulting in a reduction in FRET efficiency. This assay achieves a 39.7 aM detection limit for target miRNA-21, approximately 37-fold higher than that of the conventional assay (1.5 fM). Moreover, quantitative detection is possible in a wide range from 1 aM to 1pM within 90min with high sequence specificity. We demonstrate the assay with the detection of target miRNA-21 in total RNA extracted from MCF-7 cancer cells.

Electrochemical determination of Pb2+ based on DNAzyme-triggered rolling circle amplification and DNA-templated silver nanoclusters amplification strategy

Lead ion (Pb2+) is one of the most widespread heavy metal contaminants that pose detrimental impact on environment and human health. An electrochemical biosensor was developed for detection of Pb2+ based on rolling circle amplification (RCA) of Pb2+-dependent DNAzyme and DNA-templated silver nanoclusters (DNA-AgNCs) mediated amplification strategy. The Pb2+-dependent DNAzyme (catalytic strand) hybridized with substrate strand to form a duplex structure. In the presence of Pb2+, DNAzyme was activated and cleaved substrate strand into two fragments at the “rA” site, releasing the enzyme strand as a padlock probe for subsequent RCA. The padlock probe hybridized with primer strand to form a circular template, which triggered RCA reaction in the presence of phi29 DNA polymerase and dNTPs. The resulted RCA product hybridized with DNA-AgNCs, catalyzing H2O2 to generate enhanced electrochemical signal. The electrochemical signal was proportional to the concentration of Pb2+ in the range of 1 pM−10 μM with a detection limit of 0.3 pM (S/N = 3). The proposed strategy exhibits highly sensitive and selective for Pb2+ analysis, providing potential application in real water samples and environment analysis.

Rolling Circle Amplification on a Bead: Improving the Detection Time for a Magnetic Bioassay.

Detection of pathogens has become increasingly important, especially in the face of outbreaks and epidemics all over the world. Nucleic acid detection techniques provide a solid base to detect and identify pathogens. In recent years, magnetic sensors and magnetic labels have become of more interest due to their simplicity of use, low cost, and versatility. In this work, we have used the isothermal DNA amplification technique of rolling circle amplification (RCA) in combination with oligo-functionalized magnetic nanoparticles. Detection of RCA products takes place through specific binding between magnetic nanoparticles and RCA products. Upon binding, the relaxation frequency of the nanoparticle changes. This change was measured using an AC susceptometer. We showcase that the RCA time can be reduced for a quicker assay when performing the RCA on the surface of micrometer-sized beads, which consequently increases the hydrodynamic volume of the RCA products. This, in turn, increases the Brownian relaxation frequency shift of the nanoparticles upon binding. We performed optimization work to determine the ideal quantity of micrometer-sized particles, oligo-functionalized nanoparticles, and the amplification time of the RCA. We show that the detection of 0.75 fmol of preamplification synthetic target is possible with only 20 min of amplification time. Finally, we showcase the high specificity of the assay, as the functionalized nanoparticles are unable to bind to amplified DNA that does not match their labels. Overall, this paves the way for a simple bioassay that can be used without expensive laboratory equipment for detection of pathogens in outbreak settings and clinics around the world.

Structure-switching locked hairpin triggered rolling circle amplification for ochratoxin A (OTA) detection by ICP-MS

Ochratoxin A (OTA) is usually found in the contaminated cereal food that its trace concentration would cause severe carcinogenicity and neurotoxicity. Herein, a recognition-triggered structure switching-based element spectrometric assay was proposed for ultrasensitive and highly specific detection of OTA. OTA aptamer was first adapted into a primer for the formation of a locked hairpin (LHP), and followed by attachment on the surface of magnetic beads (MBs). In the presence of OTA, the binding event between OTA molecule and its aptamer induced the liberation of the primer for that is otherwise unable to spontaneously structure-switching. For the rolling circle amplification (RCA), thereby a dumbbell structure was designed as the template (DT). The hybridization of the dumbbell template with free primer initiates the rolling circle amplification along with the production of repeated sequences. To obtain the signal output, the DNA-AuNP tag was prepared which contained the complementary sequences with RCA products. Thus, each tag can hybridize with the repeated sequences derived from the RCA, enabling the formation of detectable signal by the following ICP-MS measurement. Such design can translate a single recognition event into numerous AuNPs, enabling amplified detection of OTA. Taking advantage of this principle, the OTA in real sample (AFP, PSA) can be quantified at femtomolar concentration and it can be expanded into application in food safety inspection.

First Report of Tomato Leaf Curl Bangladesh Virus Associated with Leaf Yellowing Symptoms in Lablab purpureus in Bangladesh

First Report of Tomato Leaf Curl Bangladesh Virus Associated with Leaf Yellowing Symptoms in <i>Lablab purpureus</i> in Bangladesh

Photoresponsive DNA-Modified Magnetic Bead-Assisted Rolling Circle Amplification-Driven Visual Photothermal Sensing of Escherichia coli.

The development of facile, reliable, and accurate assays for pathogenic bacteria is critical to environmental pollution surveillance, traceability analysis, prevention, and control. Here, we proposed a rolling circle amplification (RCA) strategy-driven visual photothermal smartphone-based biosensor for achieving highly sensitive monitoring of Escherichia coli (E. coli) in environmental media. In this design, E. coli could specifically bind with its recognition aptamer for initiating the RCA process on a magnetic bead (MB). Owing to the cleaving of UV irradiation toward photoresponsive DNA on MB, the RCA products were released to further hybridize with near-infrared excited CuxS-modified DNA probes. As a result, the photothermal signal was enhanced by RCA, while the background was decreased by UV irradiation and magnetic separation. The correspondingly generated photothermal signals were unambiguously recorded on a smartphone, allowing for an E. coli assay with a low detection limit of 1.8 CFU/mL among the broad linear range from 5.0 to 5.0 × 105 CFU/mL. Significantly, this proposed biosensor has been successfully applied to monitor the fouling levels of E. coli in spring water samples with acceptable results. This study holds great prospects by integrating a RCA-driven photothermal amplification strategy into a smartphone to develop accurate, reliable, and efficient analytical platforms against pathogenic bacteria pollutions for safeguarding environmental health.

Accurate detection of lung cancer-related microRNA through CRISPR/Cas9-assisted garland rolling circle amplification.

MicroRNA (miRNA) is reported to be closely related to a variety of pathophysiological processes for carcinoma and considered a potential biomarker for the diagnosis of lung cancer with brain metastasis. However, developing an accurate and sensitive miRNA detection method has proven to be a challenge. The aim of the present study was to integrate the advantages of rolling circle amplification (RCA), clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nucleases 9 (Cas9), and catalytic hairpin assembly (CHA) technologies to develop an miRNA detection method. In the present study, we developed a novel approach for the sensitive and accurate detection of miRNA through integrating garland RCA and CRISPR/Cas9-assisted signal generation. In this method, target miRNA cyclized dumbbell padlock and triggered the RCA process to form long single-stranded DNA products with a repeated hairpin structure. Double-stranded DNA sequences (dsDNA) were formed with the addition of complementary sequences. With the assistance of the Cas9 enzyme for specific recognition and cleavage of formed dsDNA, RCA products were disassembled into hairpin probes. The generated hairpin probe could be unfolded by target miRNA to initiate the CHA process for signal generation. Through integration of the RCA and CHA processes, the method demonstrated favorable detection performance. The correlation equation between the signal and concentration of target miRNA was determined to be Y=312.3 × lgC + 2108, with a high correlation coefficient of 0.9786. The approach also exhibited high selectivity to the mismatched miRNAs. Our method could be used in the screening, diagnosis, and prognosis of multiple diseases without complicated thermal cycling instrumentation.

Triple-hairpin mediated feedback system based on rolling circle amplification for enhanced imaging and detection of intracellular microRNA

It remains challenging to develop highly sensitive biosensing methods for the detection of short, non-coding RNA, such as microRNAs (miRNAs) that were expressed at low levels in biological environments. Here we report a triple-hairpin mediated feedback system based on rolling circle amplification (THF-RCA) for enhanced imaging and detection of intracellular miRNA. THF-RCA contains two RCA-based tandem reactions, Cycle I and Cycle II. Cycle I is initiated by a target-primed RCA reaction to produce the RCA product that can successively open HP1, HP2, and HP3. The target-analogue embedded in HP3 can be activated accompanied by the dissolving of HP3 and go on to initiate another RCA reaction, named Cycle II. Subsequently, the RCA product of Cycle II can initiate a new round of Cycle I in a feedback manner. Overall, the triple-hairpin structures serve as the pivot to power the THF-RCA reaction continues autonomously. THF-RCA based cycle system can be used to detect miR-21 down to 1 fM, and target miRNA can be identified obviously from mutation versions and no-target miRNAs. Notably, the THF-RCA based strategy allows not only for in vitro detection of miRNAs without reverse transcription, but also for intracellular imaging of miRNA with higher sensitivity compared with miRNA-FISH technology. THF-RCA based strategy is expected to provide new doors for developing novel signal amplification technologies and biosensors applied in early tumor diagnosis and early treatment.

Sensitive Methicillin-Resistant Staphylococcus Aureus (MRSA) Detection Using Gold Nanoparticle-Based Satellite Network With Potential For Early Diagnosis of Diabetes-Related Infection

Background: As pathogenic bacteria account for the leading cause of diabetes-related infections, sensitive detection of bacteria from clinical samples has attracted abundant attention. Methods: We propose an innovative DNA-AuNP-based sensing system that integrates low-speed centrifugal bacteria isolation, detection and protein analysis. In the method, RCA (rolling circle amplification) is utilized to produce a long-ssDNA (single-strand DNA), which can form a hairpin structure comprising repeats of functional domains, such as PBP2a aptamer. When aptamers bind to target bacteria, the hairpin structure in the RCA product changes its conformation, exposing the AuNP binding sequence. As a result, the probe on the surface of AuNP hybridizes with AuNP binding sequence in RCA product by strand displacement reaction, releasing the fluorescent-labeled complementary probe as the detection signal. The simultaneous formation of the bacteria-DNAAuNP satellite network enables the isolation of target bacteria by low-speed centrifugation. Results: Eventually, we applied the method for MRSA (methicillin-resistant Staphylococcus aureus) detection and obtained a favorable detection performance with a limit of detection of 275 cfu/μL. Conclusion: We believe the method has potential application in the early diagnosis of diabetesrelated infections.

Mixed infection of a new begomovirus, Jatropha leaf curl Guntur virus and recombinant/chimeric jatropha leaf curl Gujarat virus in Jatropha gossypiifolia.

The Jatropha gossypiifolia plant showing the severe leaf curl symptoms grown in the borders of chilli fields in Guntur, Andhra Pradesh, India was collected. The infection of begomovirus was detected using the degenerate primers followed by rolling circle amplification (RCA). The RCA products digested with KpnI and EcoRI showing the unit length of the begomovirus genome were cloned in pUC19 and sequenced to obtain the complete begomoviral genome. The sequence information of DNA-A of the two clones GuWC10 contained 2794 nt (MZ217773) and an incomplete genome GuWC3 with 2337 nt (MZ217772). The BLAST analysis of GuWC3 and GuWC10 sequence showed 85·57% identity with jatropha leaf curl Gujarat virus (JLCGV) and 82·68% identity with croton yellow vein mosaic virus (CroYVMV), respectively. The sequence analysis also showed that the GuWC10 clone had a 177 bp recombinant/chimeric sequence of JLCGV while the other region containing 2611 bp showed 92·63% identity with papaya leaf curl virus (PaLCuV/PK). However, the global alignment of the GuWC10 sequence showed a maximum of 80·60% identity with croton yellow vein virus (CroYVV) (FN645902), CroYVMV (JN817516) and PaLCuV/PK (KY978407). The second clone GuWC3 although shorter in length had recombinant sequences of JLCGV, jatropha leaf curl virus (JLCuV/ND) and okra enation leaf curl virus (OELCuV). The nucleotide sequence identity among the GuWC10 and GuWC3 was 71·9%. The phylogenetic analysis placed both the viral strains in the same clade located between PaLCuV/PK and JLCuV clades. According to the ICTV species demarcation criteria of 91% DNA-A sequence identity, the present isolate was considered as a new species of begomovirus and the name Jatropha leaf curl Guntur virus was proposed. This is the first report of a new begomovirus species infecting J. gossypiifolia and the study also reports a mixed infection of Jatropha leaf curl Guntur virus with a recombinant/chimeric JLCGV in the host J. gossypiifolia. Present study suggests the role of weed Jatropha in harbouring begomoviruses and probable source for viral recombination.

107P Ultra-sensitive monitoring mutations in cancer patients using superRCA mutation detection assays

107P Ultra-sensitive monitoring mutations in cancer patients using superRCA mutation detection assays

Molecular detection of disease-causing agent infecting Malvastrum coromandelianum L. in Bahawalpur District Punjab Pakistan

Weeds are common ancestors of modern crops. They are critical source of genes for resistance to diseases, pests and stresses such as drought and extreme environmental conditions. The wild relatives led to improve resistance to wheat curl mite, late blight in potato and to grassy stunt disease in rice. Agricultural pathogens are infecting economically important cash crops and weeds as well. Malvastrum coromandelianum is highly invasive weed specie of family Malvaceae. It is distributed all over the world in all climatic condition of tropical, sub-tropical and temperate region. The Malvaceae family is economically important as cotton (Gossipium hirsutem) belongs to it. M. coromandelianum has ethnobotanical importance and is extensively applied in the treatment of different diseases in south Punjab of Pakistan. It has been observed that there is a specific disease that affects the leaves of M. coromandelianum. In this study, we investigated the disease-causing agents (viruses and bacteria) at molecular level. Molecular investigation includes DNA isolation of unknown pathogens, verification through agarose gel electrophoresis, for identification of bacterial pathogens, 16s RNA primers were applied, and for viral pathogen using specific primers and RCA (rolling circle amplification) product as template. PCR was carried out for the amplification of full-length genomes. Manifestation of 1.4 kb bands showed the presence of the alpha and beta satellite of begomoviruse. Two types of begomoviruses, Malvestrum yellow vain younnan virus (MaYVYV) and Malvastrum yellow vain virus (MaYVV) was confirmed in this weed. The intermediate host Bemisia tabaci is the most common for the begomovirus transmission in crops and weeds. Intercrops and trap-crops techniques decrease the rate of whitefly population. Cultural methods, use of chemicals and biocontrol are useful practices to eliminate whitefly from field.

CRISPR/Cas9 mediated triple signal amplification platform for high selective and sensitive detection of single base mutations

Single base mutations detection is crucial for the diagnosis and treatment of cancer. However, the current methods with poor selectivity and sensitivity required large instruments, which are difficult to meet clinical demands. Herein, we develop a CRISPR/Cas9 based visual colorimetric platform to specifically detect all single base mutations. In this strategy, the Recombinase Polymerase Amplification (RPA) was firstly used to amplify the target, and introduced the PAM site in the target DNA sequence by designing the point mutation primer, thus achieving detection for all single base mutations by the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated specific recognition. With the help of CRISPR/Cas9 system, those RPA products can release single strand DNA to hybridize with the padlock probe and trigger rolling circle amplification (RCA). Based on the magnetic separation, HRP-gold nanoparticles complex (hGNPs) and biotin modified probe (Bio-probe) were further used to achieve enhanced visual variations assay by hybridizing with RCA products. Benefiting from the RPA assisted triple signal amplification, this method not only showed enhanced sensitivity with a limit of detection (LOD) as low as 0.2 fM and 0.01% of KRAS-G12D mutation percentage, but the specificity against KRAS-G12D mutation also be synergistically enhanced by combining the CRISPR/Cas9-mediated specific recognition with the specific T4 ligation reaction of RCA system. Furthermore, this system has been successfully used to visually detect genome in serum, suggesting its great potential for point-of-care diagnosis in clinical.

First Report of Squash leaf curl China virus Associated with Mosaic and Mild Leaf Curl Disease of Pumpkin in Bangladesh

First Report of <i>Squash leaf curl China virus</i> Associated with Mosaic and Mild Leaf Curl Disease of Pumpkin in Bangladesh

Isothermal Rolling Circle Amplification and Lanthanide‐Based FRET for Femtomolar Quantification of MicroRNA

AbstractInvited for this month′s cover are the collaborating groups of Prof. Niko Hildebrandt at Université de Rouen Normandie, France, and Dr. Pierre Busson at Gustave Roussy, France. The cover picture shows a Rolling Circle Amplification (RCA) product formed from a single microRNA and its detection via lanthanide (Tb and Eu) based multicolor FRET. More information can be found in the Research Article by N. Hildebrandt and co‐workers.

Simple Detection of DNA Methyltransferase with an Integrated Padlock Probe.

DNA methyltransferases (MTases) can be regarded as biomarkers, as demonstrated by many studies on genetic diseases. Many researchers have developed biosensors to detect the activity of DNA MTases, and nucleic acid amplification, which need other probe assistance, is often used to improve the sensitivity of DNA MTases. However, there is no integrated probe that incorporates substrates and template and primer for detecting DNA MTases activity. Herein, we first designed a padlock probe (PP) to detect DNA MTases, which combines target detection with rolling circle amplification (RCA) without purification or other probe assistance. As the substrate of MTase, the PP was methylated and defended against HpaII, lambda exonuclease, and ExoI cleavage, as well as digestion, by adding MTase and the undestroyed PP started RCA. Thus, the fluorescent signal was capable of being rapidly detected after adding SYBRTM Gold to the RCA products. This method has a detection limit of approximately 0.0404 U/mL, and the linear range was 0.5–110 U/mL for M.SssI. Moreover, complex biological environment assays present prospects for possible application in intricacy environments. In addition, the designed detection system can also screen drugs or inhibitors for MTases.

Rolling circle amplification of begomoviral DNA from a single nucleus isolated by laser dissection microscopy

Laser dissection microscopy (LDM) is a method for isolating organelles, a specific cell or cells/tissue of interest from microscopic regions with the help of a laser. Here we describe a LDM-based isolation of begomovirus infected Nicotiana benthamiana epidermal cells and nuclei, in combination with a fast method to prepare non-fixed leaf epidermal samples for LDM. The bipartite Abutilon mosaic virus (AbMV) was used in which the coat protein gene of DNA A was deleted and replaced by the open reading frame (ORF) coding for the green fluorescent protein (GFP, accession: U87624), agro-infiltrated together with DNA B, to visualize infected cells. GFP expressing epidermal cells or nuclei were isolated by LDM with the MMi Cellcut system and viral circular DNA was amplified by rolling circle amplification (RCA). Subsequently, the RCA product was incubated with the restriction enzymes BamHI and PstI and restriction fragments were separated on an agarose gel to prove presence of the viral genome. It was shown that even a single-isolated nucleus harbored enough material to produce a sufficient restriction fragment pattern to identify a begomovirus infected cell/nucleus.

Single Particle Inductively Coupled Plasma Mass Spectrometry-Based Homogeneous Detection of HBV DNA with Rolling Circle Amplification-Induced Gold Nanoparticle Agglomeration.

A highly sensitive and simple method based on rolling circle amplification (RCA) and single particle inductively coupled plasma mass spectrometry (spICP-MS) was proposed for the homogeneous detection of hepatitis B virus (HBV) deoxyribonucleic acid (DNA). In the presence of target DNA, long ssDNA possessing a large number of repeating sequence units was generated by RCA. DNA-labeled AuNP probes assembled into long chains based on complementary base pairing, further aggregating into large particles. Small Au NPs hardly produced pulse signals in spICP-MS; obvious pulse signals appeared in spICP-MS after the agglomeration of Au NPs caused by the addition of RCA products and spermidine. On the basis of this, the homogeneous detection of target DNA was realized by spICP-MS with high sensitivity. Under optimal conditions, the proposed method exhibited a good linear relationship between the frequency of the pulse signal of Au in spICP-MS and the concentration of target HBV DNA in the range of 10-2000 fmol L-1 (R = 0.997), the limit of detection was 5.1 fmol L-1, and the relative standard deviation was 3.7-6.8%. Recoveries of 94.2-108% were obtained for target DNA in spiked serum samples, demonstrating a good matrix tolerance ability for the method.

Engineering an endonuclease-assisted rolling circle amplification synergistically catalyzing hairpin assembly mediated fluorescence platform for miR-21 detection

As one of the earliest miRNAs discovered in the human genome, miRNA-21 can provide vital information for the early diagnosis, drug treatment, and prognosis of cancers. Herein, we construct a fast, time-saving fluorescence detection system for miRNA-21 detection by coalescing the improved endonuclease-mediated rolling circle amplification with catalytic hairpin assembly (RCA-NESA-CHA). Firstly, the target miRNA cyclized the padlock, initiating rolling circle amplification (RCA) and extending a long-concatenated DNA. The modified Protector bonded with the long-strand DNA to generate an endonuclease-specific site and trigger the nicking process. Finally, DNA products with repetitive sequences not only recombined with the padlock to reactivate a new recycle of RCA but also triggered the catalytic hairpin assembly to form the H1–H2 complex, realizing the cooperative amplification of the signal. In this system, RCA-NESA and CHA were integrated into one step, which essentially simplifies the sensing process. Moreover, the introduction of the Protector would inhibit the extension reaction caused by the combination of the padlock and the RCA products, slowing down the non-specific reaction time and improving the sensitivity of the fluorescence detection system. Under the optimal experimental conditions, the fluorescence system achieved a limit-of-detection of 0.025 amol miR-21 in a 40 μL sample and successfully applied to miR-21 detection in serum samples from breast cancer patients, showing good agreement with the results of RT-PCR, which exhibited great potential in biomedical research and clinical diagnosis.

Sensitive detection of exosomes by gold nanoparticles labeling inductively coupled plasma mass spectrometry based on cholesterol recognition and rolling circle amplification

Exosomes, potential biomarkers for liquid biopsy, provide a promising opportunity for early cancer diagnosis in a noninvasive manner. However, direct analysis of exosomes in complicated biological samples is still challenging. Herein, a cholesterol recognition assay was proposed for detection of exosomes by gold nanoparticles (Au NPs) labeling inductively coupled plasma mass spectrometry. Specifically, exosomes were captured by anti-CD63, and then cholesterol-modified rolling circle amplification product was inserted into the membrane of exosomes for signal amplification. Simultaneously, Au NPs-labelled DNA was prepared for labeling and read-out signal. The proposed method achieved the quantification of the exosome concentration in a range of 10-106 particles/μL with a limit of detection of 1.5 particles/μL. The method was applied for the direct detection of exosomes in human serum in a purification-free way, and the analytical result coincided with that obtained by nanoparticle tracking analysis. Real sample analysis manifested that the exosome concentration in the serum of oral squamous cell carcinoma patients was higher than that in the healthy volunteers’ serum. The developed method is sensitive, selective, accurate and can be used for the direct detection of exosomes in human serum, providing a new opportunity for exosome-based liquid biopsy in early cancer diagnosis.