scholarly journals Isothermal digital detection of microRNA using background-free molecular circuit

2019 ◽  
Author(s):  
Guillaume Gines ◽  
Roberta Menezes ◽  
Kaori Nara ◽  
Anne-Sophie Kirstetter ◽  
Valérie Taly ◽  
...  
Keyword(s):  
Regulation Of Gene Expression ◽  
Isothermal Amplification ◽  
Absorption Mechanism ◽  
Digital Readout ◽  
Molecular Programming ◽  
Bodily Fluids ◽  
Specific Amplification ◽  
Femtomolar Concentration ◽  
Accurate Quantification ◽  
Digital Counting

AbstractMicroRNA, a class of transcripts involved in the regulation of gene expression, are emerging as promising disease-specific biomarkers accessible from tissues or bodily fluids. However, their accurate quantification from biological samples remains challenging. We report a sensitive and quantitative microRNA method using an isothermal amplification chemistry adapted to a droplet digital readout. Building on molecular programming concepts, we design DNA circuit that converts, threshold, amplifies and report the presence of a specific microRNA, down to the femtomolar concentration. Using a leak-absorption mechanism, we were able to suppress non-specific amplification, classically encountered in other exponential amplification reactions. As a result, we demonstrate that this isothermal amplification scheme is adapted to digital counting of microRNA: by partitioning the reaction mixture into water-in-oil droplets, resulting in single microRNA encapsulation and amplification, the method provides absolute target quantification. The modularity of our approach enables to repurpose the assay for various microRNA sequences.

scholarly journals Isothermal digital detection of microRNAs using background-free molecular circuit

2020 ◽  
Vol 6 (4) ◽  
pp. eaay5952 ◽  
Author(s):  
Guillaume Gines ◽  
Roberta Menezes ◽  
Kaori Nara ◽  
Anne-Sophie Kirstetter ◽  
Valerie Taly ◽  
...  
Keyword(s):  
Regulation Of Gene Expression ◽  
Isothermal Amplification ◽  
Absorption Mechanism ◽  
Molecular Programming ◽  
Bodily Fluids ◽  
Microrna Detection ◽  
Nonspecific Amplification ◽  
Femtomolar Concentration ◽  
Accurate Quantification ◽  
Digital Counting

MicroRNAs, a class of transcripts involved in the regulation of gene expression, are emerging as promising disease-specific biomarkers accessible from tissues or bodily fluids. However, their accurate quantification from biological samples remains challenging. We report a sensitive and quantitative microRNA detection method using an isothermal amplification chemistry adapted to a droplet digital readout. Building on molecular programming concepts, we design a DNA circuit that converts, thresholds, amplifies, and reports the presence of a specific microRNA, down to the femtomolar concentration. Using a leak absorption mechanism, we were able to suppress nonspecific amplification, classically encountered in other exponential amplification reactions. As a result, we demonstrate that this isothermal amplification scheme is adapted to digital counting of microRNAs: By partitioning the reaction mixture into water-in-oil droplets, resulting in single microRNA encapsulation and amplification, the method provides absolute target quantification. The modularity of our approach enables to repurpose the assay for various microRNA sequences.

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.

scholarly journals rEXPAR: an isothermal amplification scheme that is robust to autocatalytic parasites

2019 ◽  
Author(s):  
Georg Urtel ◽  
Jean-Christophe Galas ◽  
André Estevez-Torres
Keyword(s):  
Programming Languages ◽  
Dna Polymerase ◽  
Reaction Network ◽  
Isothermal Amplification ◽  
Sequence Design ◽  
Molecular Programming ◽  
Nicking Enzyme

ABSTRACTIn the absence of DNA, a solution containing the four deoxynucleotidetriphosphates (dNTPs), a DNA polymerase and a nicking enzyme generates a self-replicating mixture of DNA species called parasite. Parasites are problematic in template-based isothermal amplification schemes such as EXPAR, as well as in related molecular programming languages, such as the PEN DNA toolbox. Here we show that the nicking enzyme Nb.BssSI allows to change the sequence design of EXPAR templates in a way that prevents the formation of parasites when dATP is removed from the solution. This method allows to make the EXPAR reaction robust to parasite contamination, a common feature in the laboratory, while keeping it compatible with PEN programs, which we demonstrate by engineering a parasite-proof bistable reaction network.

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 Real-time kinetics and high-resolution melt curves in single-molecule digital LAMP to differentiate and study specific and non-specific amplification

2020 ◽  
Vol 48 (7) ◽  
pp. e42-e42 ◽  
Author(s):  
Justin C Rolando ◽  
Erik Jue ◽  
Jacob T Barlow ◽  
Rustem F Ismagilov
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Single Molecule ◽  
High Throughput Sequencing ◽  
Limit Of Detection ◽  
Isothermal Amplification ◽  
Rapid Diagnostics ◽  
High Resolution Melt ◽  
Specific Amplification ◽  
The Impact

Abstract Isothermal amplification assays, such as loop-mediated isothermal amplification (LAMP), show great utility for the development of rapid diagnostics for infectious diseases because they have high sensitivity, pathogen-specificity and potential for implementation at the point of care. However, elimination of non-specific amplification remains a key challenge for the optimization of LAMP assays. Here, using chlamydia DNA as a clinically relevant target and high-throughput sequencing as an analytical tool, we investigate a potential mechanism of non-specific amplification. We then develop a real-time digital LAMP (dLAMP) with high-resolution melting temperature (HRM) analysis and use this single-molecule approach to analyze approximately 1.2 million amplification events. We show that single-molecule HRM provides insight into specific and non-specific amplification in LAMP that are difficult to deduce from bulk measurements. We use real-time dLAMP with HRM to evaluate differences between polymerase enzymes, the impact of assay parameters (e.g. time, rate or florescence intensity), and the effect background human DNA. By differentiating true and false positives, HRM enables determination of the optimal assay and analysis parameters that leads to the lowest limit of detection (LOD) in a digital isothermal amplification assay.

scholarly journals Loop-Mediated Isothermal Amplification Allows Rapid, Simple and Accurate Molecular Diagnosis of Human Cutaneous and Visceral Leishmaniasis Caused by Leishmania infantum When Compared to PCR

2021 ◽  
Vol 9 (3) ◽  
pp. 610
Author(s):  
Ana Victoria Ibarra-Meneses ◽  
Carmen Chicharro ◽  
Carmen Sánchez ◽  
Emilia García ◽  
Sheila Ortega ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Real Time ◽  
Parasite Load ◽  
Isothermal Amplification ◽  
Excellent Correlation ◽  
Loop Mediated Isothermal Amplification ◽  
Real Time Quantitative Pcr ◽  
Specific Amplification ◽  
Polymerase Chain ◽  
Eiken Chemical

Loop-mediated isothermal amplification allows the rapid, sensitive and specific amplification of DNA without complex and expensive equipment. We compared the diagnostic performance of Loopamp™ Leishmania Detection Kit (Eiken Chemical Co., Ltd., Tokyo, Japan) with conventional and real-time polymerase chain reaction (PCR) for human cutaneous and visceral leishmaniasis caused by L. infantum. A total of 230 DNA samples from cutaneous (CL) and visceral (VL) leishmaniasis cases and controls from Spain, characterized by Leishmania nested PCR (LnPCR) were tested by: (i) the Loopamp™ Leishmania Detection Kit (Loopamp), run on Genie III real-time fluorimeter (OptiGene, UK); and (ii) real-time quantitative PCR (qPCR). The Loopamp test returned 98.8% (95% confidence interval—CI: 96.0–100.00) sensitivity and specificity of 97.7% (95% CI: 92.2–100) on VL samples, and 100% (95% CI: 99.1–100) sensitivity and 100.0% (95% CI: 98.8–100.0) specificity on CL samples. The Loopamp time-to-positivity (Tp) obtained by real-time fluorimetry showed excellent concordance (C = 97.91%) and strong correlation (r = 0.799) with qPCR’s cycle threshold (Ct). The performance of Loopamp is comparable to that of LnPCR and qPCR in the diagnosis of cutaneous and visceral leishmaniasis due to L. infantum. The excellent correlation between the Tp and Ct should be further investigated to determine the accuracy of Loopamp to quantify parasite load in tissues.

scholarly journals The role of miRNA in selected tumors development: Potential use in diagnostics

2021 ◽  
Vol 75 ◽  
pp. 1-11
Author(s):  
Patrycja Paciorek ◽  
Mariusz Żuberek ◽  
Agnieszka Grzelak
Keyword(s):  
Gene Expression ◽  
Mirna Expression ◽  
Regulation Of Gene Expression ◽  
Regulatory Function ◽  
Cancer Information ◽  
Bodily Fluids ◽  
Experimental Therapies ◽  
Oncological Diagnostics ◽  
Potential Use

MicroRNAs (miRNAs) are small ribonucleic acid molecules that, although not translated, perform an important regulatory function in eukaryotic cells. Their physiological function is to maintain cell homeostasis. Impaired miRNA expression can cause the development of many diseases including cancer. MiRNA biological activity is based on inhibiting the formation of proteins, including oncogenic and anti-oncogenic proteins. Mutations at the coding sites for such miRNAs can lead to overproduction or reduction of the production of the above-mentioned proteins. The discovery of miRNAs and understanding their role in the cell opened new ways for diagnosing cancer. Therefore, changes in the level of relevant miRNAs in the bloodstream or other bodily fluids can be a diagnostic marker of disease. Oncological diagnostics could be based on examining the patient’s miRNA profile and comparing it with previously developed profiles of miRNAs changes associated with the occurrence of a given type of cancer. Information on changes in miRNA profiles that are key to regulating gene expression associated with tumorigenic processes could contribute to the development of experimental therapies based on restoring the original level of miRNA in cells and thereby restoring normal regulation of gene expression. New methods of silencing and enabling miRNA expression may, in the future, result in effective therapeutic solutions.

scholarly journals Rapid and sensitive detection of Salmonella species targeting the hilA gene using a loop-mediated isothermal amplification assay

2021 ◽  
Vol 19 (3) ◽  
pp. e30
Author(s):  
Jiyon Chu ◽  
Juyoun Shin ◽  
Shinseok Kang ◽  
Sun Shin ◽  
Yeun-Jun Chung
Keyword(s):  
Point Of Care ◽  
Lamp Assay ◽  
Conventional Polymerase Chain Reaction ◽  
Isothermal Amplification ◽  
Sensitive Detection ◽  
Conventional Pcr ◽  
Pcr Assay ◽  
Loop Mediated Isothermal Amplification ◽  
Amplification Assay ◽  
Specific Amplification

Salmonella species are among the major pathogens that cause foodborne illness outbreaks. In this study, we aimed to develop a loop-mediated isothermal amplification (LAMP) assay for the rapid and sensitive detection of Salmonella species. We designed LAMP primers targeting the hilA gene as a universal marker of Salmonella species. A total of seven Salmonella species strains and 11 non-Salmonella pathogen strains from eight different genera were used in this study. All Salmonella strains showed positive amplification signals with the Salmonella LAMP assay; however, there was no non-specific amplification signal for the non-Salmonella strains. The detection limit was 100 femtograms (20 copies per reaction), which was ~1,000 times more sensitive than the detection limits of the conventional polymerase chain reaction (PCR) assay (100 pg). The reaction time for a positive amplification signal was less than 20 minutes, which was less than one-third the time taken while using conventional PCR. In conclusion, our Salmonella LAMP assay accurately detected Salmonella species with a higher degree of sensitivity and greater rapidity than the conventional PCR assay, and it may be suitable for point-of-care testing in the field.

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