scholarly journals RNase H-dependent amplification improves the accuracy of rolling circle amplification combined with loop-mediated isothermal amplification (RCA-LAMP)

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11851
Author(s):  
Takema Hasegawa ◽  
Diana Hapsari ◽  
Hitoshi Iwahashi
Keyword(s):  
Detection Limit ◽  
Rolling Circle Amplification ◽  
Dna Amplification ◽  
Rnase H ◽  
Isothermal Amplification ◽  
Rolling Circle ◽  
Loop Mediated Isothermal Amplification ◽  
Specific Amplification ◽  
Negative Condition ◽  
Rnase H2

The hybrid method upon combining rolling circle amplification and loop-mediated isothermal amplification (RCA-LAMP) was developed to quantify very small amount of different type of RNAs, such as miRNAs. RCA-LAMP can help detect short sequences through padlock probe (PLP) circularization and exhibit powerful DNA amplification. However, one of the factors that determines the detection limit of RCA-LAMP is non-specific amplification. In this study, we improved the accuracy of RCA-LAMP through applying RNase H-dependent PCR (rhPCR) technology. In this method, the non-specific amplification was suppressed by using the rh primer, which is designed through blocking the modification at the 3′end to stop DNA polymerase reaction and replacing the 6th DNA molecule from the end with RNA using RNase H2 enzyme. Traditional RCA-LAMP amplified the non-specific amplicons from linear PLP without a targeting reaction, while RCA-LAMP with rh primer and RNase H2 suppressed the non-specific amplification. Conversely, we identified the risk posed upon conducting PLP cyclization reaction using Splint R ligase in the RNA-targeting step that occurred even in the RNA-negative condition, which is another factor determining the detection limit of RCA-LAMP. Therefore, this study contributes in improving the accuracy of RNA quantification using RCA-LAMP.

The influence of temperature on the multimeric products formation by strand-displacement DNA polymerases in the conditions of isothermal amplification

Biomics ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 469-474
Author(s):  
A.R. Gilvanov ◽  
A.R. Sakhabutdinova ◽  
R.R. Garafutdinov
Keyword(s):  
Rolling Circle Amplification ◽  
Dna Polymerases ◽  
Good Alternative ◽  
Clinical Diagnostics ◽  
Isothermal Amplification ◽  
Strand Displacement ◽  
Rolling Circle ◽  
Influence Of Temperature ◽  
Influence Of Temperature On ◽  
Specific Amplification

The nucleic acids amplification is one of the key methods for molecular biology research and clinical diagnostics. The isothermal amplification methods, for example rolling circle amplification, are a good alternative for a widely spread polymerase chain reaction. Strand-displacement DNA polymerases are required for isothermal amplification. In this work, we studied the influence of temperature on the formation of specific and non-specific amplification products by 9°Nm, Vent exo-, Hemo KlenTaq DNA polymerases during rolling circle amplification. The temperature values for the most effective formation of non-specific products and specific concatemeric products were determined. The obtained data will allow the development of more specific isothermal amplification methods with DNA polymerases used.

scholarly journals Rolling Circle and Loop Mediated Isothermal Amplification Strategy for Ultrasensitive miRNA Detection

Separations ◽  
2021 ◽  
Vol 8 (10) ◽  
pp. 166
Author(s):  
Zheng Cao ◽  
Xianfeng Jiang ◽  
Guizhou Xiao ◽  
Mingcheng Xu ◽  
Hui Liu ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Clinical Sample ◽  
Rolling Circle Amplification ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Rolling Circle ◽  
Loop Mediated Isothermal Amplification ◽  
Limit Of Quantitation ◽  
Mirna Detection ◽  
Amplification Strategy

Rolling circle amplification (RCA) and loop mediated isothermal amplification (LAMP) were combined to establish the rolling circle and loop mediated isothermal amplification (RC-LAMP) method for miRNA detection. With the participation of Bst 2.0 DNA Polymerase, the method enabled RCA and LAMP amplification to occur simultaneously without thermal cycling. The limit of detection of RC-LAMP was 500 amol/L, which is comparable to previously reported amplification strategies. Moreover, its upper limit of quantitation was higher and showed a stronger resistance to matrix interference. Therefore, it is possible to detect low concentrations of miRNA in samples by increasing the total RNA added. Owing to its facile detection mode and simple operation, this method has great potential in clinical sample detection.

Ferromagnetic Resonance Biosensor for Homogeneous and Volumetric Detection of DNA

2017 ◽  
Author(s):  
Bo Tian ◽  
Peter Svedlindh ◽  
Mattias Strömberg ◽  
Erik Wetterskog
Keyword(s):  
Magnetic Nanoparticles ◽  
Ferromagnetic Resonance ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Rolling Circle Amplification ◽  
Resonance Field ◽  
Isothermal Amplification ◽  
Detection Sensitivity ◽  
Serum Samples ◽  
Rolling Circle

In this work, we demonstrate for the first time, a ferromagnetic resonance (FMR) based homogeneous and volumetric biosensor for magnetic label detection. Two different isothermal amplification methods, <i>i.e.</i>, rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP) are adopted and combined with a standard electron paramagnetic resonance (EPR) spectrometer for FMR biosensing. For RCA-based FMR biosensor, binding of RCA products of a synthetic Vibrio cholerae target DNA sequence gives rise to the formation of aggregates of magnetic nanoparticles. Immobilization of nanoparticles within the aggregates leads to a decrease of the net anisotropy of the system and a concomitant increase of the resonance field. A limit of detection of 1 pM is obtained with an average coefficient of variation of 0.16%, which is superior to the performance of other reported RCA-based magnetic biosensors. For LAMP-based sensing, a synthetic Zika virus target oligonucleotide is amplified and detected in 20% serum samples. Immobilization of magnetic nanoparticles is induced by their co-precipitation with Mg<sub>2</sub>P<sub>2</sub>O<sub>7</sub> (a by-product of LAMP) and provides a detection sensitivity of 100 aM. The fast measurement, high sensitivity and miniaturization potential of the proposed FMR biosensing technology makes it a promising candidate for designing future point-of-care devices.<br>

scholarly journals Biosensors Based on Isothermal DNA Amplification for Bacterial Detection in Food Safety and Environmental Monitoring

Sensors ◽  
2021 ◽  
Vol 21 (2) ◽  
pp. 602
Author(s):  
Sandra Leonardo ◽  
Anna Toldrà ◽  
Mònica Campàs
Keyword(s):  
Food Safety ◽  
Environmental Monitoring ◽  
Rolling Circle Amplification ◽  
Dna Amplification ◽  
Isothermal Amplification ◽  
In Situ Monitoring ◽  
Strand Displacement ◽  
Strand Displacement Amplification ◽  
Bacterial Detection ◽  
Efficient Detection

The easy and rapid spread of bacterial contamination and the risk it poses to human health makes evident the need for analytical methods alternative to conventional time-consuming laboratory-based techniques for bacterial detection. To tackle this demand, biosensors based on isothermal DNA amplification methods have emerged, which avoid the need for thermal cycling, thus facilitating their integration into small and low-cost devices for in situ monitoring. This review focuses on the breakthroughs made on biosensors based on isothermal amplification methods for the detection of bacteria in the field of food safety and environmental monitoring. Optical and electrochemical biosensors based on loop mediated isothermal amplification (LAMP), rolling circle amplification (RCA), recombinase polymerase amplification (RPA), helicase dependent amplification (HDA), strand displacement amplification (SDA), and isothermal strand displacement polymerisation (ISDPR) are described, and an overview of their current advantages and limitations is provided. Although further efforts are required to harness the potential of these emerging analytical techniques, the coalescence of the different isothermal amplification techniques with the wide variety of biosensing detection strategies provides multiple possibilities for the efficient detection of bacteria far beyond the laboratory bench.

Mitigating the Non-Specific Amplification in RCA: Assessment of the Role of Ligation and Exonuclease Digestion in Circular DNA Preparation

2021 ◽  
Author(s):  
Vandana Kuttappan Nair ◽  
Chandrika Sharma ◽  
Mrittika Sengupta ◽  
Souradyuti Ghosh
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
False Positive ◽  
Rolling Circle Amplification ◽  
Circular Dna ◽  
Rolling Circle ◽  
Specific Amplification ◽  
Exonuclease Digestion ◽  
Sticky End

<b>Layman Summary: </b>Rolling circle amplification (RCA) is a popular and extensively used bioanalytical tool. Like any nucleic acid amplifications, non-specific amplification may occur in it and risk generating false positive readouts. The work described in the manuscript investigates non-specific amplification in RCA as a function of ligation and exonuclease digestion assays during the synthesis of circular DNA. In particular, it investigates and compares the role of three different ligation techniques, namely splint-padlock ligation, cohesive end (sticky end ligation), and self-annealing ligation. In addition, it also probes the role of single exonuclease vs dual exonuclease digestions. We employed real time fluorescence to quantify the effect of these factors. Finally, our work hypothesizes the possible origins of non-specific amplification in RCA.

scholarly journals Reverse Transcription Loop-Mediated Isothermal Amplification for the Detection of Porcine Reproductive and Respiratory Syndrome Virus

2009 ◽  
Vol 21 (3) ◽  
pp. 350-354 ◽  
Author(s):  
Albert Rovira ◽  
Juan Abrahante ◽  
Michael Murtaugh ◽  
Muñoz-Zanzi Claudia
Keyword(s):  
Reverse Transcription ◽  
Limit Of Detection ◽  
Restriction Analysis ◽  
Dna Amplification ◽  
Lamp Reaction ◽  
Isothermal Amplification ◽  
Respiratory Syndrome Virus ◽  
Rt Pcr ◽  
Serum Samples ◽  
Loop Mediated Isothermal Amplification

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen of swine. The objective of the current study is to investigate the feasibility of using reverse transcription loop-mediated isothermal amplification (RT-LAMP) for the detection of PRRSV. The RT-LAMP is a recently described DNA amplification technique reported to be simple, inexpensive, fast, and accurate. The RT-LAMP reaction was set up using 2 sets of primers that were designed to detect North American and European strains of PRRSV and performed successfully in a simple heat block. The specificity of the amplified product was demonstrated by restriction analysis. The RT-LAMP was able to detect 5 different PRRSV isolates. However, the limit of detection ranged between 10 2 and 10 4 50% tissue culture infective dose/ml. The RT-LAMP was further evaluated using serum samples from animals of known infection status. The ability of RT-LAMP to detect PRRSV in serum from acutely infected animals was evaluated with 114 serum samples from 18 experimentally inoculated boars. Forty-nine of these samples tested positive by RT-LAMP, while 94 were positive by reverse transcription polymerase chain reaction (RT-PCR). The diagnostic specificity, evaluated with 100 known negative serum samples, was estimated as 99%. The feasibility of RT-LAMP to detect PRRSV was demonstrated in the current study. The RT-LAMP reaction could be performed in just 1 hr with a simple and inexpensive heat block. However, the sensitivity of this technique was significantly lower than that of RT-PCR.

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