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An inter-platform repeatability study investigating real-time amplification of plasmid DNA
Carol E Donald, Fizza Qureshi, Malcolm J Burns, Marcia J Holden, Joseph R Blasic, Alison J Woolford
BMC Biotechnology , 2005, DOI: 10.1186/1472-6750-5-15
Abstract: Statistical analysis of the data arising from three different real-time platforms was conducted in order to assess inter-platform repeatability. On three consecutive days two PCR reaction mixes were used on each of the three amplification platforms – the LightCycler?, ABI PRISM? 7700 and Rotor Gene 3000?. Real-time PCR amplification using a fluorogenic 5' exonuclease assay was performed in triplicate on negative controls and DNA plasmid dilutions of 108–102 copies to give a total of 24 reactions per PCR experiment.The results of the statistical analyses indicated that the platform with the most precise repeatability was the ABI PRISM? 7700 when coupled with the FastStart PCR reaction mix. It was also found that there was no obvious relationship between plasmid copy number and repeatability. An ANOVA approach identified the factors that significantly affected the results, in descending order of magnitude, as: plasmid copy number, platform, PCR reaction mix and day (on which the experiment was performed).In order to deliver useful, informative genetic tests, standardisation of real-time PCR detection platforms to provide repeatable, reliable results is warranted. In addition, a better understanding of inter-assay and intra-assay repeatability is required.A diverse range of applications that impact on clinical diagnosis and prognosis rely on the real-time detection and quantification of Polymerase Chain Reaction products such as bacterial diagnostics [1,2], viral load [3-6], cancerous cell burden [7,8] and parasite quantification [9]. Each particular assay dictates the requirement for instrument capacity, data reproducibility and run length times. There is an expansive collection of different fluorescent chemistries and instruments available for real-time amplification reactions [10-12]. These platforms represent a technological advance from the traditional thermal cycler and typically offer faster, smaller (reduced volume) and more (high-throughput) reactions. Whilst
Low Cost Extraction and Isothermal Amplification of DNA for Infectious Diarrhea Diagnosis  [PDF]
Shichu Huang, Jaephil Do, Madhumita Mahalanabis, Andy Fan, Lei Zhao, Lisa Jepeal, Satish K. Singh, Catherine M. Klapperich
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0060059
Abstract: In order to counter the common perception that molecular diagnostics are too complicated to work in low resource settings, we have performed a difficult sample preparation and DNA amplification protocol using instrumentation designed to be operated without wall or battery power. In this work we have combined a nearly electricity-free nucleic acid extraction process with an electricity-free isothermal amplification assay to detect the presence of Clostridium difficile (C. difficile) DNA in the stool of infected patients. We used helicase-dependent isothermal amplification (HDA) to amplify the DNA in a low-cost, thermoplastic reaction chip heated with a pair of commercially available toe warmers, while using a simple Styrofoam insulator. DNA was extracted from known positive and negative stool samples. The DNA extraction protocol utilized an air pressure driven solid phase extraction device run using a standard bicycle pump. The simple heater setup required no electricity or battery and was capable of maintaining the temperature at 65°C±2°C for 55 min, suitable for repeatable HDA amplification. Experiments were performed to explore the adaptability of the system for use in a range of ambient conditions. When compared to a traditional centrifuge extraction protocol and a laboratory thermocycler, this disposable, no power platform achieved approximately the same lower limit of detection (1.25×10?2 pg of C. difficile DNA) while requiring much less raw material and a fraction of the lab infrastructure and cost. This proof of concept study could greatly impact the accessibility of molecular assays for applications in global health.
Isothermal Amplification Using a Chemical Heating Device for Point-of-Care Detection of HIV-1  [PDF]
Kelly A. Curtis, Donna L. Rudolph, Irene Nejad, Jered Singleton, Andy Beddoe, Bernhard Weigl, Paul LaBarre, S. Michele Owen
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0031432
Abstract: Background To date, the use of traditional nucleic acid amplification tests (NAAT) for detection of HIV-1 DNA or RNA has been restricted to laboratory settings due to time, equipment, and technical expertise requirements. The availability of a rapid NAAT with applicability for resource-limited or point-of-care (POC) settings would fill a great need in HIV diagnostics, allowing for timely diagnosis or confirmation of infection status, as well as facilitating the diagnosis of acute infection, screening and evaluation of infants born to HIV-infected mothers. Isothermal amplification methods, such as reverse-transcription, loop-mediated isothermal amplification (RT-LAMP), exhibit characteristics that are ideal for POC settings, since they are typically quicker, easier to perform, and allow for integration into low-tech, portable heating devices. Methodology/Significant Findings In this study, we evaluated the HIV-1 RT-LAMP assay using portable, non-instrumented nucleic acid amplification (NINA) heating devices that generate heat from the exothermic reaction of calcium oxide and water. The NINA heating devices exhibited stable temperatures throughout the amplification reaction and consistent amplification results between three separate devices and a thermalcycler. The performance of the NINA heaters was validated using whole blood specimens from HIV-1 infected patients. Conclusion The RT-LAMP isothermal amplification method used in conjunction with a chemical heating device provides a portable, rapid and robust NAAT platform that has the potential to facilitate HIV-1 testing in resource-limited settings and POC.
Microfluidic Extreme PCR: <1 Minute DNA Amplification in a Thin Film Disposable  [PDF]
James M. Trauba, Carl T. Wittwer
Journal of Biomedical Science and Engineering (JBiSE) , 2017, DOI: 10.4236/jbise.2017.105017
Abstract: Point-of-care diagnostic testing using PCR requires a device that is fast, economical, and practical. Sub-minute amplification has been demonstrated using high concentrations of primers and polymerase in glass capillaries, but its platform is limited to research use. A system using heated copper blocks to clamp a microfluidic flow-through PCR card fabricated from thin film polycarbonate was modeled, fabricated, and tested. Models show that fluid flowing through a thin-film device clamped between temperature-controlled copper blocks equilibrates to a temperature change in 250 milliseconds. A 2-step, 35 cycle PCR with 1.06 second cycles specifically amplified a 69-base pair fragment from a 450-base pair synthetic DNA template of random sequence with the same performance as the glass capillary system. This system demonstrates the feasibility of <1 minute PCR in an inexpensive, disposable sample container.
Dynamics and Control of DNA Sequence Amplification  [PDF]
Karthikeyan Marimuthu,Raj Chakrabarti
Quantitative Biology , 2014, DOI: 10.1063/1.4899053
Abstract: DNA amplification is the process of replication of a specified DNA sequence \emph{in vitro} through time-dependent manipulation of its external environment. A theoretical framework for determination of the optimal dynamic operating conditions of DNA amplification reactions, for any specified amplification objective, is presented based on first-principles biophysical modeling and control theory. Amplification of DNA is formulated as a problem in control theory with optimal solutions that can differ considerably from strategies typically used in practice. Using the Polymerase Chain Reaction (PCR) as an example, sequence-dependent biophysical models for DNA amplification are cast as control systems, wherein the dynamics of the reaction are controlled by a manipulated input variable. Using these control systems, we demonstrate that there exists an optimal temperature cycling strategy for geometric amplification of any DNA sequence and formulate optimal control problems that can be used to derive the optimal temperature profile. Strategies for the optimal synthesis of the DNA amplification control trajectory are proposed. Analogous methods can be used to formulate control problems for more advanced amplification objectives corresponding to the design of new types of DNA amplification reactions.
Real-Time Fluorescence Loop Mediated Isothermal Amplification for the Detection of Acinetobacter baumannii  [PDF]
Qinqin Wang, Yanbin Zhou, Shaoli Li, Chao Zhuo, Siqi Xu, Lixia Huang, Ling Yang, Kang Liao
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0066406
Abstract: Background Detection of Acinetobacter baumannii has been relying primarily on bacterial culture that often fails to return useful results in time. Although DNA-based assays are more sensitive than bacterial culture in detecting the pathogen, the molecular results are often inconsistent and challenged by doubts on false positives, such as those due to system- and environment-derived contaminations. In addition, these molecular tools require expensive laboratory instruments. Therefore, establishing molecular tools for field use require simpler molecular platforms. The loop-mediated isothermal amplification method is relatively simple and can be improved for better use in a routine clinical bacteriology laboratory. A simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in the same platform has been developed in recent years. This method is referred to as real-time loop-mediated isothermal amplification. In this study, we attempted to utilize this method for rapid detection of A. baumannii. Methodology and Significant Findings Species-specific primers were designed to test the utility of this method. Clinical samples of A. baumannii were used to determine the sensitivity and specificity of this system compared to bacterial culture and a polymerase chain reaction method. All positive samples isolated from sputum were confirmed to be the species of Acinetobacter by 16S rRNA gene sequencing. The RealAmp method was found to be simpler and allowed real-time detection of DNA amplification, and could distinguish A. baumannii from Acinetobacter calcoaceticus and Acinetobacter genomic species 3. DNA was extracted by simple boiling method. Compared to bacterial culture, the sensitivity and specificity of RealAmp in detecting A. baumannii was 98.9% and 75.0%, respectively. Conclusion The RealAmp assay only requires a single unit, and the assay positivity can be verified by visual inspection. Therefore, this assay has great potential of field use as a molecular tool for detection of A. baumannii.
Silicene as a new ultrafast DNA sequencing device  [PDF]
Rodrigo G. Amorim,Ralph H. Scheicher
Physics , 2014, DOI: 10.1088/0957-4484/26/15/154002
Abstract: Silicene, a hexagonal buckled 2-D allotrope of silicon, shows potential as a platform for numerous new applications, and may allow for easier integration with existing silicon-based microelectronics than graphene. Here, we show that silicene could function as an ultrafast electrical DNA sequencing device. We investigated the stability of this novel nano-bio system, its electronic properties and the pronounced effects on the transverse electronic transport, i.e., changes in the transmission and the conductance caused by adsorption of each nucleobase, explored by us through the non-equilibrium Green's function method. Intriguingly, despite the relatively weak interaction between nucleobases and silicene, significant changes in the transmittance at zero bias are predicted by us, in particular for the two nucleobases cytosine and guanine. Our findings suggest that silicene could be utilized as an integrated-circuit biosensor as part of a lab-on-a-chip device for DNA sequencing.
Detection of Nucleic Acid Targets Using Ramified Rolling Circle DNA Amplification: A Single Nucleotide Polymorphism Assay Model  [PDF]
James H. Smith, Thomas P. Beals
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0065053
Abstract: Background Isothermal amplification methods provide alternatives to PCR that may be preferable for some nucleic acid target detection tasks. Among current isothermal target detection methods, ramified rolling circle amplification (RAM) of single-stranded DNA circles that are formed by ligation of linear DNA probes (C-probes or padlock probes) offers a unique target detection system by linked primers and a simple amplification system that is unconstrained by the target’s sequence context. Earlier implementations of RAM-based target detection were reported to be limited by background noise, due in part to unligated C-probe in the amplification reaction. We show here that a target-detection system using a biotinylated target-capture probe together with automated bead-handling reduces or eliminates background amplification noise. We demonstrate the system’s performance by detection of a single-nucleotide polymorphism in human genomic DNA. Methodology Target detection by RAM entails hybridization and ligation of a C-probe, followed by amplification and RAM signal detection. We evaluated RAM target detection in genomic DNA using recognition of a human Factor V gene single nucleotide polymorphism (G1691A) as a model. Locus-specific C-probes were annealed and ligated to genomic DNAs that represent the 3 possible genotypes at this locus, then ligated C-probes were amplified by real time RAM. The majority of the steps in the assay were performed with a magnetic bead-based chemistry on an automated platform. We show that the specificity of C-probe ligation permits accurate genotyping of this polymorphism. The assay as described here eliminates some of the background noise previously described for C-probe ligation, RAM amplification assays. Conclusion The methods and results presented here show that a combination of C-probe detection, automated sample processing, and isothermal RAM amplification provide a practical approach for detecting DNA targets in complex mixtures.
DNA Circuits Based on Isothermal Constrained Loop Extension DNA Amplification  [PDF]
Maurice Margenstern,Pascal Mayer,Sergey Verlan
Computer Science , 2011,
Abstract: In this paper, we first describe the isothermal constrained loop extension DNA amplification (ICLEDA), which is a new variant of amplification combining the advantages of rolling circle amplification (RCA) and of strand displacement amplification (SDA). Then, we formalize this process in terms of the theory of formal languages and show, on the basis of this formulation, how to manage OR and AND gates. We then explain how to introduce negation, which allows us to prove that, in principle, it is possible to implement the computation of any boolean function on DNA strands using ICLEDA.
Nuclease-resistant double-stranded DNA controls or standards for hepatitis B virus nucleic acid amplification assays
Shuang Meng, Sien Zhan, Jinming Li
Virology Journal , 2009, DOI: 10.1186/1743-422x-6-226
Abstract: The obtained armored DNA was resistant to DNase I digestion and was stable, noninfectious to humans, and could be easily extracted using commercial kits. More importantly, the armored DNA may be used with all HBV DNA nucleic acid amplification assay kits.The lambda phage packaging system can be used as an excellent expression platform for armored DNA. The obtained armored DNA possessed all characteristics of an excellent positive control or standard. In addition, this armored DNA is likely to be appropriate for all commercial HBV DNA nucleic acid amplification detection kits. Thus, the constructed armored DNA can probably be used as a universal positive control or standard in HBV DNA assays.Hepatitis B virus (HBV) infection is a major public health problem. It is responsible for chronic liver disease and is a risk factor for liver cirrhosis and hepatocellular carcinoma [1-3]. Early diagnosis and measurement of viral load in patients with HBV is very helpful for the management of this disease [2].Real-time polymerase chain reaction (PCR) assays are widely used for the detection and quantification of HBV DNA in clinical samples [4-6]. Quantitative detection of HBV DNA in serum or plasma provides evidence about the level of viral replication, degree of infection, and efficacy of antiviral therapy [7-11]. Many commercial kits are available for HBV DNA quantification. Each kit uses proprietary HBV DNA standards or controls and provides results unique to that particular method. All of the HBV DNA nucleic acid amplification detection kits target the highly conserved surface antigen gene or the precore-core genes of the HBV genome [10,12-17] and the HBV DNA controls or standards come from many sources [18-20].Owing to the lack of a universal HBV DNA standard, identical blood samples yield markedly different HBV DNA levels when tested by different kits [21-23]. These inconsistent results complicate the interpretation of viral load and could influence clinicians+ decisions. T
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