Combination Of Rolling Circle Amplification Research Articles

Development of loop mediated isothermal amplification (LAMP): A new tool for rapid diagnosis of cotton leaf curl viral disease

Cotton leaf curl disease (CLCuD) ranks top among all endemic diseases transmitted by whitefly (Bemisia tabaci) affecting cotton (Gossypium hirsutum) causing severe economic losses to the cotton growers in the Indian subcontinent. For its effective management, robust tools for detection are a prerequisite and it is important to diagnose the virus titre in early stage of infection in plants as well as in the disease transmitting vector. Considering the limitations in current PCR-based techniques we have standardised rapid and sensitive Loop Mediated Isothermal Amplification (LAMP) protocol for the diagnosis of cotton leaf curl virus (CLCuV) in cotton leaves and in its transmitting vector whitefly. Perhaps, this is the first report of use of LAMP tool for rapid diagnosis of CLCuV in cotton and its transmitting vector the whitefly. Further, the colorimetric detection for diagnostic simplicity of amplified LAMP product by using different dyes lead to enhanced applicability of this technique in the field of disease diagnostics. The merit of present study is that the diagnostic failure of PCR and LAMP due to low virus titre in the infected leaf has been circumvented through the combination of rolling circle amplification (RCA) with LAMP. Thus RCA-LAMP can be an option for ultra-sensitive detection of samples with low virus titre. The potential applications of this advanced diagnostic tool in laboratory research on diagnosis of CLCuV, an important viral pathogen of cotton have been discussed.

A Convenient and Sensitive Method for Deoxynucleoside Triphosphate Quantification by the Combination of Rolling Circle Amplification and Quantitative Polymerase Chain Reaction.

Measurement of four dNTP pools is important for investigating metabolism, genome stability, and drug action. In this report, we developed a two-step method for quantitating dNTPs by the combination of rolling circle amplification (RCA) and quantitative polymerase chain reaction (qPCR). We used CircLigase to generate a single-strand DNA in circular monomeric configuration, which was then used for the first step of RCA reaction that contained three dNTPs in excess for quantification of one dNTP at limiting levels. The second step is the amplification of RCA products by qPCR, in which one primer was designed to be completely annealed with the polymeric ssDNA product but not the monomeric template DNA. Using 1 amol of the template in the assay, each dNTP from 0.02 to 2.5 pmol gave a linearity with r2 > 0.99, and the quantification was not affected by the presence of rNTPs. We further found that the preparation of biological samples for the RCA reaction required methanol and chloroform extraction. The method was so sensitive that 1 × 104 cells were sufficient for dNTP quantification with the results similar to those determined by a radio-isotope method using 2 × 105 cells. Thus, the RCA/qPCR method is convenient, cost-effective, and highly sensitive for dNTP quantification.

Genome characterization of parsley severe stunt-associated virus in Iran.

Parsley severe stunt-associated virus (PSSaV) is a recently identified nanovirus first reported in Germany. During a survey for identification of nanoviruses infecting apiaceous plants in south-eastern Iran, PSSaV was identified and characterized using a combination of rolling circle amplification (RCA) and high-throughput sequencing. Parsley plant samples were collected from vegetable production farms in Kerman province. From two symptomatic samples (39Ba and 40Ba), seven PSSaV components (DNA-C, -S, -M, -R, -N, -U1 and -U2) with two phylogenetically distinct variants of DNA-R (R1 and R2) were identified. In common with the German isolate of PSSaV, no DNA-U4 component was identified. In addition, associated alphasatellite molecules were identified in samples 39Ba [n = 6] and 40Ba [n = 5]. Sequence analyses showed that concatenated component sequences of the two Iranian PSSaVs share 97.2% nucleotide identity with each other and 82% to the German isolate. The coat proteins (CPs) of the PSSaV Iranian sequences share 97.2% amino acid identity and ~ 84% identity with that of the German isolate. Sequence and phylogenetic analyses of a total of 11 recovered alphasatellites from the two samples can be classified into the genera Fabenesatellite [n = 2], Milvetsatellite [n = 1], Mivedwarsatellite [n = 2], Subclovsatellite [n = 2], Sophoyesatellite [n = 4] in the family Alphasatellitidae. Identification of PSSaV and other nanoviruses in wild and cultivated plants in Iran reveals that nanoviruses could be causing yield reduction in crops plants in this country.

Isolation and characterization of a novel geminivirus from parsley

Fresh leaf vegetables are a significant part of the Persian food. Following a survey for identification of nanoviruses and geminivirus infecting leaf vegetables, a novel geminivirus was identified in a diseased parsley sample showing upward marginal leaf curling, marginal leaf yellowing, dwarfing and reduced leaf size in south-eastern Iran. The genome was identified through combination of rolling circle amplification (RCA) and high throughput sequencing (HTS) approaches. The full-length genome (2779 nts) of the cloned geminivirus, parsley yellow leaf curl virus (PYLCV), shares <66 % genome-wide pairwise identity with all other known geminiviruses. The PYLCV genome has six open reading frames (ORFs) and appears to be a hybrid with the virion sense encoded proteins being most similar to those of becurtoviruses and curtoviruses, whereas the complementary sense encoded proteins are most similar to those of begomoviruses. In comparison with other geminivirus encoded capsid proteins (CPs) and replication associated proteins (Reps), the CP of PYLCV shares <56 % amino acid pairwise identity whereas the Rep shares <73 % amino acid pairwise identity. To demonstrate the pathogenicity of the geminivirus, a partial dimer infectious clone was constructed and used to agro-infect parsley as well as Nicotiana benthamiana, turnip, radish and tomato. The agro-inoculation resulted in infection with symptoms in 83.7 % (82/98) of the tested plant. Based on the similarity of the CP encoded by PYLCV to those of becurtoviruses and curtoviruses, it is likely that leafhoppers may be the primary transmission vector.

Rolling Circle Amplification-Based Polyvalent Molecular Beacon Probe-Assisted Signal Amplification Strategies for Sensitive Detection of B16 Cells.

We developed a simple and sensitive signal amplification method for the detection of B16 cells based on the combination of rolling circle amplification (RCA) and molecular beacons (MBs). A long-chain structure of DNA synthesized by RCA was used to turn on aptamer-based MBs. Because of the multiple complementary repeat units, the RCA scaffold hybridized tens or hundreds of MBs to form polyvalent aptamer probes. The unfold ability and the fluorescence intensity of MBs were both improved by RCA, as compared to short single chains. The cell experiment results demonstrated that RCA-based polyvalent MBs were significantly more effective than monovalent MBs in targeting B16 cells and signal sensitivity because of their multivalent effects. The establishment of this strategy would provide a powerful platform for early clinical diagnostics of cancer cells.

Characterization and genetic diversity of Dioscorea bacilliform viruses present in a Pacific yam germplasm collection

AbstractYam is an important food staple in many Pacific Island countries, but its production is limited by several factors including virus infection. The presence of viruses also hinders the exploitation of the crop by restricting the international dissemination of yam germplasm due to quarantine concerns. In this study, a total of 224 yam accessions present in the Pacific germplasm collection at SPC‐CePaCT, Fiji, were tested for the presence of badnaviruses using a combination of rolling circle amplification (RCA) and sequencing. Of these, 35 tested positive for either Dioscorea bacilliform AL virus (DBALV) or Dioscorea bacilliform AL virus 2 (DBALV2). DBALV was only detected in samples from Vanuatu and Tonga, while DBALV2 was restricted to Papua New Guinean samples. Ten complete genome sequences of DBALV and DBALV2 were obtained and pairwise comparison of the partial reverse transcriptase/ribonuclease H (RT/RNase H)‐coding sequence revealed identities ranging from 89% to 100% and 92% to 100%, respectively. The results suggest that DBALV and DBALV2 are the prevalent badnavirus species infecting yams in the Pacific region.

A fluorometric lead(II) assay by using a DNA dendrimer as a carrier for the immobilization of the signal probe.

A DNA dendrimer was constructed by combination of rolling circle amplification (RCA) and hybridization chain reaction (HCR). A fluorescence resonance energy transfer (FRET) pair consisting of 6-carboxyfluorescein (FAM) and 5-carboxytetramethylrhodamine (TAMRA) was then created as a signaling probe for the ultrasensitive detection of Pb2+. Firstly, the DNAzyme released by Pb2+-induced recycling amplification as a primer induces RCA to open two different hairpins for generating repeated "Y" structures. Next, two other hairpins (labeled with FAM and TAMRA, respectively) are opened by the "Y" structures to trigger the HCR. As a result, a DNA dendrimer is generated. It is high loading with FRET pair and also promotes FRET pair mutually close to produce a remarkable FRET signal. Hence, ultrasensitive detection of Pb2+ is accomplished by measurement of the ratio of the yellow fluorescence of TAMRA (peakingat 588nm) and the green fluorescence of FAM (at 525nm). The method works in the 0.001 to 10nM Pb2+ concentration range and has a 0.3 pM detection limit. It was applied to determination of intracellular Pb2+ with convincing performance. Graphical abstractSchematic representation of novel DNA dendrimer produced by the combination of rolling circle amplification (RCA) and hybridization chain reaction (HCR) for immobilization of the fluorescence resonance energy transfer (FRET) pair as signal probe forsensitive determination of Pb2+.

A T7 exonuclease-RCA dual amplification system for high-sensitivity and high-selectivity analysis of microRNA

A novel combination of rolling circle amplification and T7 exonuclease-assisted amplification to achieve highly sensitive and selective detection of microRNA molecules was developed.

Rolling circle extension-actuated loop-mediated isothermal amplification (RCA-LAMP) for ultrasensitive detection of microRNAs

Rolling circle amplification (RCA) is an elegant and well-recognized isothermal nucleic acid amplification mechanism that has been widely used for the detection of various kinds of genetic biomarkers. However, traditional RCA is a linear signal amplifying mechanism so that the amplification efficiency is generally not satisfactory. Herein, we rationally combine RCA with efficient loop-mediated isothermal amplification (LAMP) to establish a rapid and ultrasensitive RCA-LAMP method for the detection of microRNAs (miRNAs). In the RCA-LAMP, the target let-7a miRNA can directly template the ligation of a padlock probe to trigger RCA reaction, producing long and tandem amplification products. Only such RCA-produced long DNA repeats can act as the template to generate a lot of double stem-loop DNAs with functional sequences, which are the essential starting materials to initiate subsequent LAMP. Finally, the products of LAMP reaction, the amount of which is dependent on the initial miRNA dosage, can be fluorescently monitored in a real-time manner. Through the combination of ligation-mediated RCA with LAMP, the amplification efficiency and the detection sensitivity has been significantly improved. As a result, even 10 aM miRNA target can be clearly and accurately detectable. Despite the excellent analytical performance for miRNA analysis, compared with conventional RCA-based miRNA assays, the combination of RCA with LAMP does not introduce any additional reaction steps or sample transfer operations. Both the RCA and LAMP are fulfilled in a single step. Therefore, this facile and ultrasensitive RCA-LAMP assay provides a new promising tool for miRNA analysis and can be extended to the detection of various kinds of genetic biomarkers.

Silica bead-based microfluidic device with integrated photodiodes for the rapid capture and detection of rolling circle amplification products in the femtomolar range

The rapid and sensitive detection of specific nucleic acid sequences at the point-of-care (PoC) is becoming increasingly in demand for a variety of emergent biomedical applications ranging from infectious disease diagnostics to the screening of antimicrobial resistance. To meet such demand, considerable efforts have been invested towards the development of portable and integrated analytical devices combining microfluidics with miniaturized signal transducers.Here, we demonstrate the combination of rolling circle amplification (RCA)-based nucleic acid amplification with an on-chip size-selective trapping of amplicons on silica beads (~8 nL capture chamber) coupled with a thin-film photodiode (200 × 200 µm area) fluorescence readout. Parameters such as the flow rate of the amplicon solution and trapping time were optimized as well as the photodiode measurement settings, providing minimum detection limits below 0.5 fM of targeted nucleic acids and requiring only 5 μL of pre-amplified sample. Finally, we evaluated the analytical performance of our approach by benchmarking it against a commercial instrument for RCA product (RCP) quantification and further investigated the effect of the number of RCA cycles and elongation times (ranging from 10 to 120 min). Moreover, we provide a demonstration of the application for diagnostic purposes by detecting RNA from influenza and Ebola viruses, thus highlighting its suitability for integrated PoC systems.

Rolling circle amplification integrated with suspension bead array for ultrasensitive multiplex immunodetection of tumor markers

Multiplex detection of ultra-low abundant tumor markers is extremely important for early diagnosis and therapy evaluation. Herein, an ultrasensitive multiplex immunoassay was developed by combination of rolling circle amplification (RCA) and suspension bead array (SBA) technology. Based on a conventional sandwich-type immunoreaction on beads, the detection antibodies were conjugated with DNA primers, so RCA could be implemented to generate long-stranded DNA with abundant repeated sequences allowing for hybridization with fluorochrome-labeled oligonucleotide probes. Thus the fluorescence signal of immunocomplexes on the encoded beads can be greatly enhanced. Using the as-developed immuno-RCA suspension bead array (iRCA-SBA), simultaneous analysis of multiple tumor markers was achieved with the limits of detection of 3.1 pg/mL (∼0.1 pM) for prostate specific antigen (PSA), 9.1 pg/mL (∼50 fM) for carcinoembryonic antigen (CEA), and 0.66 pg/mL (∼9 fM) for α-fetoprotein (AFP), which are two to three orders of magnitude lower than those obtained by the conventional SBA method. The dynamic range were 4.5, 4.7, and 5.5 orders of magnitude for PSA, CEA, and AFP, respectively. Tests on clinical serum samples demonstrate that the tumor marker concentrations measured by the newly developed iRCA-SBA assay agreed well with those obtained by the conventional SBA method. These results indicate that the iRCA-SBA assay significantly increased the detection sensitivity and dynamic range without sacrificing the reliability and accuracy of conventional SBA. Upon the integration with iRCA, SBA could find more applications in the detection of low abundance protein biomarkers for early diagnosis of cancer and other diseases.

RCA-enhanced multifunctional molecule beacon-based strand-displacement amplification for sensitive microRNA detection

MicroRNAs (miRNAs) are regarded as promising cancer-related biomarkers. Here, we developed a simple, sensitive and specific fluorescence method based on the combination of rolling circle amplification (RCA) and multifunctional molecular beacon-based strand-displacement amplification (MMB-SDA) for the amplification detection of let-7a miRNA. Specifically, a multifunctional molecular beacon (MMB) was proposed to execute several functions, including the signal reporter, primer and polymerization template. To achieve RCA reaction, 3′ and 5′ terminal bases of padlock probe were designed as the recognition regions capable of hybridizing with let-7a miRNA. Moreover, this miRNA could serve as the primer to trigger RCA reaction after ligation, producing tens and hundreds of tandemly repeated copies of cycle. Subsequently, the RCA product hybridized with MMB and initiated the repetitive SDA reaction, opening a significant amount of MMBs and causing an amplified signal. As a result, let-7a miRNA can be specifically detected down to 51 pM, and the single-base difference between let-7a miRNAs was easily detected. The potential application was demonstrated via evaluating the let-7a miRNA level in real samples (total RNA extracted from HeLa cells). Therefore, the developed sensing strategy would provide a powerful platform for early clinical diagnostics.

Generation of Large-Scale DNA Hydrogels with Excellent Blood and Cell Compatibility.

Hemocompatibility and cytocompatibility of biomaterials codetermine the success of tissue engineering applications. DNA, the natural component of our cells, is an auspicious biomaterial for the generation of designable scaffolds with tailorable characteristics. In this study, a combination of rolling circle amplification and multiprimed chain amplification is used to generate hydrogels at centimeter scale consisting solely of DNA. Using an in vitro rotation model and fresh human blood, the reaction of the hemostatic system on DNA hydrogels is analyzed. The measurements of hemolysis, platelets activation, and the activation of the complement, coagulation, and neutrophils using enzyme-linked immunosorbent assays demonstrate excellent hemocompatibility. In addition, the cytocompatibility of the DNA hydrogels is tested by indirect contact (agar diffusion tests) and material extract experiments with L929 murine fibroblasts according to the ISO 10993-5 specifications and no negative impact on the cell viability is detected. These results indicate the promising potential of DNA hydrogels as biomaterials for versatile applications in the field of regenerative medicine.

Development of rolling circle amplification based surface-enhanced Raman spectroscopy method for 35S promoter gene detection

In this study, we developed the genetically modified organism detection method by using the combination of rolling circle amplification (RCA) and surface-enhanced Raman spectroscopy (SERS). An oligonucleotide probe which is specific for 35S DNA promoter target was immobilised onto the gold slide and a RCA reaction was performed. A self-assembled monolayer was formed on gold nanorods using 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and the second probe of the 35S DNA promoter target was immobilised on the activated gold coated slide surfaces. Probes on the nanoparticles were hybridised with the target oligonucleotide. Quantification of the target concentration was performed via SERS spectra of DTNB on the nanorods. SERS spectra of target molecules were enhanced through the RCA reaction and the detection limit was found to be 6.3fM. The sensitivity of the developed RCA–SERS method was compared with another method which had been performed without using RCA reaction, and the detection limit was found to be 0.1pM. The correlation between the target concentration and the SERS signal was found to be linear, within the range of 1pM to 10nM for the traditional assay and 100fM to 100nM for the RCA assay. For the developed RCA–SERS assay, the specificity tests were performed using the 35S promoter of Bt-176 maize gene. It was found out that the developed RCA–SERS sandwich assay method is quite sensitive, selective and specific for target sequences in model and real systems.

Circomics of Cuban geminiviruses reveals the first alpha-satellite DNA in the Caribbean.

Circomics (circular DNA genomics), the combination of rolling circle amplification (RCA), restriction fragment length polymorphism (RFLP) analysis and pyro-sequencing, has been used recently to identify geminiviruses with high efficiency and low costs. Circular DNAs associated with Cuban geminiviruses were characterised by RCA/RFLP analysis and 454 sequencing of two batches of DNA amplified from selected plant samples as well as individual cloning and Sanger sequencing of DNA components and compared to other geminiviral DNAs by phylogenetic analysis. Cuban geminiviruses that were closely related to each other challenged the circomics approach. Ten geminiviral components and one alpha-satellite DNA were determined and compared to three geminiviral components obtained by conventional cloning. New strains of Sida yellow mottle virus (SiYMoV), tomato yellow distortion leaf virus (ToYDLV), Sida golden mosaic Florida virus (SiGMFV) and Sida golden mosaic Liguanea virus (SiGMLV) are described with host plant species being classified by molecular PCR-based bar coding. A new virus species is named Peristrophe mosaic virus. The first alpha-satellite found in Middle America establishes the New World branch of these elements which are related to nanoviruses and were previously thought to be restricted to the Old World. In conclusion, circomics is efficient for complex infections and closely related viruses to detected unexpected viral DNAs, but may need some scrutinisation by direct sequencing and cloning of individual components for certain cases.

Novel myco-like DNA viruses discovered in the faecal matter of various animals

A wide variety of novel single-stranded DNA (ssDNA) viruses have been found in faecal matter of chimpanzees, cows, rodents, bats, badgers, foxes and pigs over the last few years. Using a combination of rolling circle amplification coupled with restriction enzyme digests based approach as well as a next generation sequencing informed approach, we have recovered fourteen full genomes of ssDNA viruses which exhibit genomic features described for members of the recently proposed gemycircularvirus group from a wide variety of mammal and bird faecal samples across New Zealand. The fourteen novel ssDNA viruses (2122–2290nt) encode two major proteins, a replication associated protein (Rep) and a capsid protein (Cp) which are bi-directionally transcribed. Interestingly, the Rep of these novel viruses are similar to gemycircularviruses detected in insects, cassava leaves, and badger faecal matter, the novel viruses share sequence similarities with the mycovirus sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) and Rep-like sequences found in fungal genomes. Pairwise sequence similarities between the 14 novel genomes with other related viral isolates (gemycircularviruses) indicated that they share greater than 55.8% genome-wide identity. Additionally, they share between 55% and 59% pairwise identity with putative novel ssDNA virus genomes recently isolated from sewage baminivirus, niminivirus and nephavirus. Based on the similarities to SsHADV-1 and Rep-like sequences found in fungal genomes, these novel gemycircularviruses may infect fungi.

Sensitive and specific HBV genomic DNA detection using RCA-based QCM biosensor

Rolling circle amplification (RCA) is an isothermal amplification technique for small, circular DNA templates with the unique property of the product localization. This research describes the application of RCA-based quartz crystal microbalance (QCM) biosensor for direct detection of hepatitis B virus (HBV) genomic DNA from clinical samples. The covalent bonding between the capture probes and the gold electrode surface guarantees that the linkage between the capture probes and the amplified RCA products is maintained during the assay. Making use of the high amplification efficiency of Phi29 DNA polymerase and the remarkable precision of Escherichia coli DNA ligase in differentiating mismatched bases at the ligation site, as low as 104copies/mL HBV genomic DNA can be detected. Experimental results show that RCA has significantly enhanced sensitivity for the target strand compared to the single-base mismatch strand. In this paper, with 60min RCA duration, the proposed sensitive detection shows over 10 times amplification of the original signals. The combination of RCA with QCM biosensor has the potential to become a successful clinical application.

Ultrasensitive Colorimetric DNA Detection using a Combination of Rolling Circle Amplification and Nicking Endonuclease‐Assisted Nanoparticle Amplification (NEANA)

A combination of rolling circle amplification and nicking endonuclease-assisted nanoparticle amplification (NEANA) is used for the rapid, colorimetric detection of DNA. The integration of rolling circle amplification into the NEANA approach allows for detection of oligonucleotides with arbitrary sequences at ultralow concentrations.

Circular DNA genomics (circomics) exemplified for geminiviruses in bean crops and weeds of northeastern Brazil

Circomics was coined to describe the combination of rolling circle amplification (RCA), restriction fragment length polymorphism (RFLP) and pyro-sequencing to investigate the genome structures of small circular DNAs. A batch procedure is described using 61 plant samples from Asia, South America and Central America which revealed 83 contig sequences of geminiviral DNA components and 4 contig sequences of DNA satellites. The usefulness of this approach is validated for the Brazilian begomoviruses, and the sequence fidelity is determined by comparing the results with those of conventional cloning and sequencing of Bolivian begomoviruses reported recently. Therefore, circomics has been proven to be a major step forward to economize costs and labor and to characterize reliably geminiviral genomes in their population structure of the quasispecies.

Double-probe signal enhancing strategy for toxin aptasensing based on rolling circle amplification

On the basis of aptamer-based rolling circle amplification (RCA) and magnetic beads (MBs), a highly sensitive electrochemical method was developed for the determination of Ochratoxin A (OTA). Initially, an amino-modified capture DNA was immobilized onto MBs for the following hybridization with an OTA aptamer and a phosphate labeled padlock DNA. In the presence of OTA, the aptamer would dissociate from the bioconjugate, and the padlock DNA would subsequently hybridize with the capture DNA to form a circular template with the aid of the T4 ligase. Next, capture DNA would act as primer to initiate a linear RCA reaction and hence generate a long tandem repeated sequences by phi29 DNA polymerase and dNTPs. Then, two quantum dots (QDs) labeled DNA probes were tagged on the resulted RCA product to indicate the OTA recognition event by electrochemical readout. This strategy, based on the novel design of OTA-mediated DNA circularization, the combination of RCA and double signal probes introduction, could detect OTA down to the level of 0.2pgmL−1 with a dynamic range spanning more than 4 orders of magnitude. The proposed approach is tested to determine OTA in red wines and shows good application potential in real samples.