Catalytic hairpin assembly (CHA) has previously proven useful as a transduction and amplification method for nucleic acid detection. However, the two hairpin substrates in a CHA circuit can ...potentially react non‐specifically even in the absence of a single‐stranded catalyst, and this non‐specific background degrades the signal‐to‐noise ratio. The introduction of mismatched base pairs that impede uncatalyzed strand exchange reactions led to a significant decrease of the background signal, while only partially damping the signal in the presence of a catalyst. Various types and lengths of mismatches were assayed by fluorimetry, and in many instances, our MismatCHA designs yielded 100‐fold increased signal‐to‐background ratios compared to a ratio of 4:1 with the perfectly matched substrates. These observations could be of general utility for the design of non‐enzymatic nucleic acid circuits.
Catalytic hairpin assembly (CHA) is a useful amplification method for the detection of nucleic acids. CHA circuits, however, have been shown to execute non‐specifically even in the absence of catalyst, which can make quantitation of lower input concentrations difficult. By introducing two mismatched bases into a specific domain on the circuit, the signal‐to‐background ratio can be improved from less than 10:1 to over 100:1.
Catalytic hairpin assembly (CHA) is an enzyme-free amplification method that has previously proven useful in amplifying and transducing signals at the terminus of nucleic acid amplification ...reactions. Here, for the first time, we engineered CHA to be thermostable from 37 to 60 °C and in consequence have generalized its application to the real-time detection of isothermal amplification reactions. CHA circuits were designed and optimized for both high- and low-temperature rolling circle amplification (RCA) and strand displacement amplification (SDA). The resulting circuits not only increased the specificity of detection but also improved the sensitivity by as much as 25- to 10000-fold over comparable real-time detection methods. These methods have been condensed into a set of general rules for the design of thermostable CHA circuits with high signals and low noise.
The Middle East respiratory syndrome coronavirus (MERS-CoV), an emerging human coronavirus, causes severe acute respiratory illness with a 35% mortality rate. In light of the recent surge in reported ...infections we have developed asymmetric five-primer reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for detection of MERS-CoV. Isothermal amplification assays will facilitate the development of portable point-of-care diagnostics that are crucial for management of emerging infections. The RT-LAMP assays are designed to amplify MERS-CoV genomic loci located within the open reading frame (ORF)1a and ORF1b genes and upstream of the E gene. Additionally we applied one-step strand displacement probes (OSD) for real-time sequence-specific verification of LAMP amplicons. Asymmetric amplification effected by incorporating a single loop primer in each assay accelerated the time-to-result of the OSD-RT-LAMP assays. The resulting assays could detect 0.02 to 0.2 plaque forming units (PFU) (5 to 50 PFU/ml) of MERS-CoV in infected cell culture supernatants within 30 to 50 min and did not cross-react with common human respiratory pathogens.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
It is inherently difficult to quantitate nucleic acid analytes with most isothermal amplification assays. We developed loop‐mediated isothermal amplification (LAMP) reactions in which competition ...between defined numbers of “false” and “true” amplicons leads to order of magnitude quantitation by a single endpoint determination. These thresholded LAMP reactions were successfully used to directly and quantitatively estimate the numbers of nucleic acids in complex biospecimens, including directly from cells and in sewage, with the values obtained closely correlating with qPCR quantitations. Thresholded LAMP reactions are amenable to endpoint readout by cell phone, unlike other methods that require continuous monitoring, and should therefore prove extremely useful in developing one‐pot reactions for point‐of‐care diagnostics without needing sophisticated material or informatics infrastructure.
Lighting the LAMP: We developed loop‐mediated isothermal amplification (LAMP) reactions transduced by oligonucleotide strand displacement (OSD) probes in which competition between defined numbers of “false” (introduced) and “true” (analyte) amplicons leads to order of magnitude analyte quantitation by a single visual endpoint determination. These LAMP‐OSD reactions directly and quantitatively estimated numbers of nucleic acids in complex biospecimens.
Abstract
Background
In response to supply shortages caused by the COVID-19 pandemic, N95 filtering facepiece respirators (FFRs or “masks”), which are typically single-use devices in healthcare ...settings, are routinely being used for prolonged periods and in some cases decontaminated under “reuse” and “extended use” policies. However, the reusability of N95 masks is limited by degradation of fit. Possible substitutes, such as KN95 masks meeting Chinese standards, frequently fail fit testing even when new. The purpose of this study was to develop an inexpensive frame for damaged and poorly fitting masks using readily available materials and 3D printing.
Results
An iterative design process yielded a mask frame consisting of two 3D printed side pieces, malleable wire links that users press against their face, and cut lengths of elastic material that go around the head to hold the frame and mask in place. Volunteers (n = 45; average BMI = 25.4), underwent qualitative fit testing with and without mask frames wearing one or more of four different brands of FFRs conforming to US N95 or Chinese KN95 standards. Masks passed qualitative fit testing in the absence of a frame at rates varying from 48 to 94 % (depending on mask model). For individuals who underwent testing using respirators with broken or defective straps, 80–100 % (average 85 %) passed fit testing with mask frames. Among individuals who failed fit testing with a KN95, ~ 50 % passed testing by using a frame.
Conclusions
Our study suggests that mask frames can prolong the lifespan of N95 and KN95 masks by serving as a substitute for broken or defective bands without adversely affecting fit. Use of frames made it possible for ~ 73 % of the test population to achieve a good fit based on qualitative and quantitative testing criteria, approaching the 85–90 % success rate observed for intact N95 masks. Frames therefore represent a simple and inexpensive way of expanding access to PPE and extending their useful life. For clinicians and institutions interested in mask frames, designs and specifications are provided without restriction for use or modification. To ensure adequate performance in clinical settings, fit testing with user-specific masks and PanFab frames is required.
To make the electrochemical DNA sensors (E-sensor) more robust and reproducible, we have now for the first time adapted the techniques of ratiometric analyses to the field of E-sensors. We did this ...via the simple expedient way of simultaneously using two redox probes: Methylene blue as the reporter of the conformational change, and ferrocene as an internal control. During the conformational transduction, only the distance between the signal probe and the electrode surface undergoes an appreciable change, while the distance between the control probe and the electrode remains relatively constant. This special design has allowed very reliable target recognition, as illustrated in this report using a human T-lymphotropic virus type I gene fragment. The standard deviation between measurements obtained using different electrodes was an order of magnitude less than that obtained using a classic E-sensor, which we prepared as a control. A limit of detection of 25.1 pM was obtained with our new system, with a single mismatch discrimination factor of 2.33 likewise being observed. Additionally, this concept had general applicability, and preliminary data of a “Signal-On” ratiometric E-sensor are also provided. Taken in concert, these results serve to validate the utility of what we believe will emerge as an easily generalized approach to oligonucleotide recognition and sensing.
Loop-mediated isothermal amplification (LAMP) of DNA is a powerful isothermal nucleic acid amplification method that can generate upward of 109 copies from less than 100 copies of template DNA within ...an hour. Unfortunately, although the amplification reactions are extremely powerful, real-time and specific detection of LAMP products remains analytically challenging. In order to both improve the specificity of LAMP detection and to make readout simpler and more reliable, we have replaced the intercalating dye typically used for monitoring in real-time fluorescence with a toehold-mediated strand exchange reaction termed one-step strand displacement (OSD). Due to the inherent sequence specificity of toehold-mediated strand exchange, the OSD reporter could successfully distinguish side products from true amplicons arising from templates corresponding to the biomedically relevant M. tuberculosis RNA polymerase (rpoB) and the melanoma-related biomarker BRAF. OSD allowed the Yes/No detection of rpoB in a complex mixture such as synthetic sputum and also demonstrated single nucleotide specificity in Yes/No detection of a mutant BRAF allele (V600E) in the presence of 20-fold more of the wild-type gene. Real-time detection of different genes in multiplex LAMP reactions also proved possible. The development of simple, readily designed, modular equivalents of TaqMan probes for isothermal amplification reactions should generally improve the applicability of these reactions and may eventually assist with the development of point-of-care tests.
Human fecal contamination of water is a public health risk. However, inadequate testing solutions frustrate timely, actionable monitoring. Bacterial culture-based methods are simple but typically ...cannot distinguish fecal host source. PCR assays can identify host sources but require expertise and infrastructure. To bridge this gap we have developed a field-ready nucleic acid diagnostic platform and rapid sample preparation methods that enable on-site identification of human fecal contamination within 80 min of sampling. Our platform relies on loop-mediated isothermal amplification (LAMP) of human-associated Bacteroides HF183 genetic markers from crude samples. Oligonucleotide strand exchange (OSD) probes reduce false positives by sequence specifically transducing LAMP amplicons into visible fluorescence that can be photographed by unmodified smartphones. Our assay can detect as few as 17 copies/ml of human-associated HF183 targets in sewage-contaminated water without cross-reaction with canine or feline feces. It performs robustly with a variety of environmental water sources and with raw sewage. We have also developed lyophilized assays and inexpensive 3D-printed devices to minimize cost and facilitate field application.
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•A field ready isothermal assay for detection of human fecal contamination in water.•DNA strand displacement probes guarantee assay specificity.•Sample-to-answer detection in minimally processed environmental samples within 80 min.•Smartphone assay readout, chemical heating and cold chain elimination reduce cost.
Strand exchange nucleic acid circuitry can be used to transduce isothermal nucleic acid amplification products into signals that can be readable on an off-the-shelf glucometer. Loop-mediated ...isothermal amplification (LAMP) is limited by the accumulation of non-specific products, but nucleic acid circuitry can be used to probe and distinguish specific amplicons. By combining this high temperature isothermal amplification method with a thermostable invertase, we can directly transduce Middle-East respiratory syndrome coronavirus and Zaire Ebolavirus templates into glucose signals, with a sensitivity as low as 20-100 copies/μl, equating to atto-molar (or low zepto-mole). Virus from cell lysates and synthetic templates could be readily amplified and detected even in sputum or saliva. An OR gate that coordinately triggered on viral amplicons further guaranteed fail-safe virus detection. The method describes has potential for accelerating point-of-care applications, in that biological samples could be applied to a transducer that would then directly interface with an off-the-shelf, approved medical device.
During the past decade, aptasensors have largely been designed on the basis of the notion that ligand-modulated equilibration between aptamer conformations could be exploited for sensing. One ...implementation of this strategy has been to denature the aptamer with an antisense oligonucleotide, wait for dissociation of the antisense oligonucleotide, and stabilize the folded, signaling conformer with a ligand. However, there is a large kinetic barrier associated with releasing the oligonucleotide from the aptamer to again obtain an active, binding conformation. If the length of the antisense oligonucleotide is decreased to make dissociation from the aptamer more favorable, higher background signals are observed. To improve the general methodology for developing aptasensors, we have developed a novel and robust strategy for aptasensor design in which an oligonucleotide kinetically competes with the ligand for binding rather than having to be released from a stable duplex. While the oligonucleotide can induce conformational change, it initially chooses between the aptamer and a molecular beacon (MB), a process that does not require a lengthy pre-equilibration. Using an anti-ricin aptamer as a starting point, we developed a “competitive” aptasensor with a measured limit of detection (LOD) of 30 nM with an optical readout and as low as 3 nM for ricin toxin A-chain (RTA) detection on an electrochemical platform.
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