Structure Of Double-stranded DNA Research Articles

Programmable Lanthanide Metal-Organic Framework for Ultra-Efficient Nucleic Acids Extraction and Interaction Analysis.

Efficient, mild, and reversible adsorption of nucleic acids onto nanomaterials represents a promising analytical approach for medical diagnosis. However, there is a scarcity of efficient and reversible nucleic acid adsorption nanomaterials. Additionally, the lack of comprehension of the molecular mechanisms governing their interactions poses significant challenges. These issues hinder the rational design and analytical applications of the nanomaterials. Herein, we propose an ultra-efficient nucleic acid affinity nanomaterial based on programmable lanthanide metal-organic frameworks (Ln-MOFs). Through experiments and density functional theory calculations, a rational design guideline for nucleic acid affinity of Ln-MOF was proposed, and a modular and flexible preparation scheme was provided. Then, Er-TPA (terephthalic acid) MOF emerged as the optimal candidate due to its pore size-independent adsorption and desorption capabilities for nucleic acids, enabling ultra-efficient adsorption (about 150% mass ratio) within 1 min. Furthermore, we elucidate the molecular-level mechanisms underlying the Ln-MOF adsorption of single- and double-stranded DNA and G4 structures. The affinity nanomaterial based on Ln-MOF exhibits robust nucleic acid extraction capability (4-fold higher than commercial reagent kits) and enables mild and reversible CRISPR/Cas9 functional regulation. This method holds significant promise for broad application in DNA/RNA liquid biopsy and gene editing, facilitating breakthroughs in analytical chemistry, pharmacy, and medical research.

The clinical characteristics of neutrophil extracellular trap in patients with severe cerebral venous sinus thrombosis and its prognostic value in acute and subacute patients

Objective: To explore the clinical characteristics of neutrophil extracellular trap (NET) in patients with severe cerebral venous sinus thrombosis (CVST) and to study their prognostic value in the acute and subacute phases. Methods: This study is a retrospective case series analysis. Clinical and pathological data of 52 patients with severe cerebral venous sinus thrombosis who underwent endovascular treatment in the Department of Neurosurgery, Tianjin Huanhu Hospital from June 2019 to June 2022 were retrospectively analyzed. There were 20 males and 32 females, with an age of (40.1±13.6) years(range:18 to 66 years). Forty-five healthy physical examinees were included in the control group. High-resolution MRI was used to stage the thrombus, with 11 cases in the acute group, 28 cases in the subacute group, and 13 cases in the chronic group. Thrombus specimens were obtained through endovascular treatment, and the fluorescence intensity of NET in peripheral blood at different time points was analyzed by immunofluorescence contrast,including the double-stranded DNA structure and adhesion protein components (citrolinated histone H3 (CitH3), myeloperoxidase-DNA complex(MPO-DNA), neutrophil elastase (NE)). The NET markers were determined by ELISA. Spearman rank correlation analysis was used to analyze the correlation between the NET markers in peripheral blood of patients with severe cerebral venous sinus thrombosis in the acute and subacute phases and the volume of venous sinus thrombus, the degree of venous sinus recanalization after treatment, and the discharge modified Rankin scale(mRS)score. The accuracy of NET markers in predicting the prognosis of patients with severe cerebral venous sinus thrombosis was analyzed by drawing receiver operating characteristic (ROC) curve and calculating the area under the curve (AUC). Results: The results of immunofluorescence staining and ELISA showed that no NET structure was formed in the peripheral blood of the control group, while CitH3, MPO-DNA and NE levels in the peripheral blood of CVST patients were increased, among which the acute stage group was the highest, followed by the subacute group, and the chronic group was the lowest. Spearman correlation analysis showed that CitH3, MPO-DNA and NE levels in peripheral blood of patients in acute group and subacute group were positively correlated with thrombus volume and mRS score at discharge (P<0.05). The levels of CitH3 and MPO-DNA in peripheral blood of patients with complete venous sinus recanalization were lower than those of patients with partial venous sinus recanalization (P<0.01). ROC curve analysis results showed that MPO-DNA and NE had no predictive ability for the prognosis of CVST patients (P values were 0.614 and 0.324, respectively), and the AUC of CitH3 was 0.800 (95%CI: 0.638~0.962, P=0.032), the best cut-off value was 13.5 μg/L, the sensitivity was 100%, and the specificity was 58.8%. Conclusions: A large number of NET are formed in patients with severe cerebral venous sinus thrombosis in acute stage. Patients with severe cerebral venous sinus thrombosis in acute stage and subacute stage with high peripheral blood NET content has a low rate of complete sinus revascularization and poor neurological function recovery after treatment.

Amodiaquine Nonspecifically Binds Double Stranded and Three-Way Junction DNA Structures.

The 4-aminoquinoline class of compounds includes the important antimalarial compounds amodiaquine and chloroquine. Despite their medicinal importance, the mode of action of these compounds is poorly understood. In a previous study we observed these compounds, as well as quinine and mefloquine, tightly bind the DNA cocaine-binding aptamer. Here, we further explore the range of nucleic acid structures bound by these compounds. To gauge a wide range of binding affinities, we used isothermal titration calorimetry to explore high affinity binding (nM to tens of μM) and NMR spectroscopy to assay weak binding biding in the hundreds of micromolar range. We find that amodiaquine tightly binds all double stranded DNA structures explored. Mefloquine binds double stranded DNA duplex molecules tightly and weakly associates with a three-way junction DNA construct. Quinine and chloroquine only weakly bind duplex DNA but do not tightly bind any of the DNA constructs explored. A simulation of the free energy of binding of these ligands to the Dickerson-Drew dodecamer resulted in an excellent agreement between the simulated and experimental free energy. These results provide new insight into the DNA binding of clinically important antimalarial compounds and may play a role in future development of new antimalarials.

CRISPR/Cas13a-triggered entropy-driven amplification for colorimetric and fluorescent dual-mode detection of microRNA

MicroRNAs (miRNAs) are crucial biomarkers for the early detection and monitoring of disease progression of chronic obstructive pulmonary disease (COPD). Herein, we have devised a method for detecting miRNA using a combination of colorimetric and graphene oxide-based fluorescent techniques. The target miRNA in our design could precisely activate the trans-cleavage activity of the CRISPR-Cas13a system. The activated Cas13a enzyme cuts the “rUrU” section in the P1 probe, generating a nicking site to induce entropy-driven amplification (EDA). One of the available EDA products has the capability to unfold the hairpin probe, thereby initiating the catalytic hairpin assembly, exposing the G-quadruplex structure, facilitating the subsequent color response. The fuel strand labeled with Cy3 successfully established a double-stranded DNA structure with DNA3, and consequently the Cy3 would not be quenched by graphene oxide (GO). The implementation of the dual-mode technique in this method yields greater benefits in terms of improving the precision and consistency of the miRNA measurements. The developed method has the capability to fluorescently measure miRNA-21 levels down to a concentration of 5.8 fM. In addition, the analysis of miRNA targets from clinical samples using this method demonstrates its promising utility in the fields of biomedical research of COPD.

Sensitive fluorescence detection based on dimeric G-quadruplex combined with enzyme-assisted solid-phase microextraction of streptomycin in honey

Streptomycin (STR), an aminoglycoside antibiotic with the potential to persist in honey and other food products, may induce allergy, toxicity and antibiotic resistance in humans. In this study, we developed a solid-phase microextraction (SPME) biosensor based on a quartz rod that was modified with double-stranded DNA structures consisting of partially complementary G-rich base DNA strand and STR aptamer. The STR isolated by SPME initially bound to the aptamer. Then the remaining double-stranded DNA structures were cleaved by the Nt.BstNBI enzyme, resulting in release of G-quadruplex dimers. The latter formed a complex with thioflain T fluorescent dye, resulting in an amplified fluorescence response. The method exhibited high sensitivity (a limit of detection of 10.84 pM), wide linear range (0.05 nM ∼ 500 nM (with determination coefficient > 0.99)), and simple operation, making it suitable and convenient for STR detection. Successful STR determination in genuine honey samples was demonstrated.

Target-triggered dual signal amplification based on HCR-enhanced nanozyme activity for the sensitive visual detection of Escherichia coli.

The detection of foodborne pathogens is crucial for food hygiene regulation and disease diagnosis. Colorimetry has become one of the main analytical methods in studying foodborne pathogens due to its advantages of visualization, low cost, simple operation, and no complex instrument. However, the low sensitivity limits its applications in early identification and on-site detection for trace analytes. In order to overcome such a limitation, herein we propose a joint strategy featuring dual signal amplification based on the hybridization chain reaction (HCR) and DNA-enhanced peroxidase-like activity of gold nanoparticles (AuNPs) for the sensitive visual detection of Escherichia coli. Target bacteria bound specifically to the aptamer domain in the capture hairpin probe, exposing the trigger domain for HCR and forming the extended double-stranded DNA (dsDNA) structures. The peroxidase-like catalytic capacity of AuNPs can be enhanced significantly by dsDNAs with the sticky ends of dsDNAs being adsorbed on AuNPs and the rigidity of dsDNAs causing the spatial regulation of AuNP concentration. The intensity of the enhancement was linearly related to the number of target bacteria. With the above strategy, the detection limit of our colorimetric method for Escherichia coli was down to 28 CFU mL-1 within a short analytical time (50 min). This study provides a new perspective for the sensitive and visual detection of early bacterial contamination in foods.

Electron/Hole Mobilities of Periodic DNA and Nucleobase Structures from Large-Scale DFT Calculations.

Electron/hole transfer mechanisms in DNA and polynucleotide structures continue to garner considerable interest as emerging charge-transport systems and molecular electronics. To shed mechanistic insight into these electronic properties, we carried out large-scale density functional theory (DFT) calculations (up to 650 atoms) to systematically analyze the structural and electron/hole transport properties of fully periodic single- and double-stranded DNA. We examined the performance of various exchange-correlation functionals (LDA, BLYP, B3LYP, and B3LYP-D) and found that single-stranded thymine (T) and cytosine (C) are predominantly hole conductors, whereas single-stranded adenine (A) and guanine (G) are better electron conductors. For double-stranded DNA structures, the periodic A-T and G-C electronic band structures undergo a significant renormalization, which causes hole transport to only occur on the A and G nucleobases. Our calculations (1) provide new benchmarks for periodic nucleobase structures using dispersion-corrected hybrid functionals with large basis sets and (2) highlight the importance of dispersion effects for obtaining accurate geometries and electron/hole mobilities in these extended systems.

Hairpin DNA-enabled ratiometric electrochemical aptasensor for detection of malathion

A hairpin DNA-enabled ratiometric electrochemical aptasensor is reported for sensitive and reliable detection of malathion (MAL). The approach employs hairpin DNA (ferrocene-labeled, Fc-hDNA) as a carrier to hybridize MAL aptamers (methylene blue-labeled, MB-Apt) to form double-stranded DNA structures on an electrode. The presence of MAL induces the removal of aptamers, and hDNA re-forms hairpin structures, causing a decrease in the oxidation current of MB (IMB) and an increase in the oxidation current of Fc (IFc). The ratiometric signal of IFc/IMB responds quantitatively to MAL concentrations. To compare analytical performances, a linear single-stranded DNA (ssDNA) is also used to construct the ssDNA-based aptasensor. We demonstrate that hairpin DNA possessing a rigid two-dimensional structure can improve the assembly efficiency of aptamers and the stability of redox probes. The approach combines the advantages of the ratiometric electrochemical method with hairpin DNA-based conformational switching probes, enabling hDNA-based aptasensor with enhanced sensitivity and reliability, offering a linear range of 0.001 to 1.0 ng mL-1. The platform was applied to detect MAL in lettuce, and the statistical analysis indicated that no significant differences were found between the developed platform and HPLC-MS.

A Fluorogenic Covalent Chromone-Based Intercalator with a Mega-Stokes Shift for Sensing DNA Hybridization

Forced intercalation (FIT) probes have proven to be a reliable, rapid, inexpensive, and accurate method for the detection and visualization of specific nucleic acid sequences. The accommodation of a rationally designed chromone-based fluorogen within a double-stranded DNA structure was investigated by UV–Vis spectrophotometry and steady-state fluorescence spectroscopy under physiological pH conditions. After selective excitation matching with a 350 nm laser, the intrinsically negligible fluorescence of the tethered electroneutral label in a single-stranded context was increased 10-fold upon duplex formation. This fluorescence enhancement was also accompanied by a mega-Stokes shift (~100 nm) that placed the emission in the cyan color range; both features are appreciable for bio-imaging purposes. In sum, its fluorogenic behavior and its marginal impact on the double helix make this dye a prospective tool for selectively sensing sequences of interest with a remarkable ON/OFF contrast.

Fluorescence off–on nanosensor based on MoS2 nanosheets and oligonucleotides for the alternative detection of mercury(II) ions or silver(I) ions

As traditional methods for detection of heavy metal pollution in water involve complex procedures and require expensive equipment, there is a great deal of interest in the development of rapid and simple methods for determining heavy metal ions in water. Here, a nanobiosensor based on molybdenum disulphide (MoS2) nanosheets and fluorophore (FAM) labeled oligonucleotides was proposed, and fluorescence spectroscopy was adopted for detection of Hg2+ or Ag+ ions in aqueous solution. The principle underlying detection by the sensor involves the formation of T-Hg2+-T or C-Ag+-C mismatches by single-stranded DNA (ssDNA) rich in thymine (T) or cytosine (C), thereby forming stable double-stranded DNA (dsDNA) structures. By exploiting the different adsorption capacity of MoS2 nanosheets for ssDNA and dsDNA, when oligonucleotides were in a single chain state, MoS2 nanosheets possessed a strong adsorption capacity for ssDNA, resulting in fluorescence quenching of FAM. After the addition of Hg2+ or Ag+, ssDNA formed double chains structure, the fluorescence recovered due to the weak adsorption capacity of MoS2 nanosheets for dsDNA. Along this line, an “off-on” mode fluorescence nanobiosensor was designed to alternatively detect these two heavy metal ions in water. The sensor showed high sensitivity and excellent selectivity for both Hg2+ and Ag+ ions, with minimum detection limits of 6.8 nM and 8.9 nM, respectively.

DNA bending facilitates the formation of Z-DNA investigated by single-molecule FRET.

Z-DNA, a noncanonical helical structure of double-stranded DNA (dsDNA), plays essential roles in diverse biological processes such as transcription regulation. Mechanical stresses on dsDNA, such as torsion and tension, could form Z-DNA more easily. However, the effect of DNA bending, one of the most common deformations of DNA, on Z-DNA formation is utterly elusive. Herein, we show that DNA bending facilitates the formation of Z-DNA, that is, Z-DNA is formed at lower Mg2+ concentration as the bending force becomes stronger. We regulated the bending force on dsDNA via D-shaped DNA nanostructures and observed the B-Z transition by single-molecule FRET. In the presence of the bending force, the B-Z transition of methylated DNA occurred at a 28-fold lower Mg2+ condition (2.8 mM) which is in the range of physiological salt concentrations. Monte Carlo simulation suggested that the base extrusion at the B-Z junction according to the B-Z transition may stabilize the Z-DNA form via releasing the bending stress on dsDNA. Our results certainly show that DNA bending force induces the formation of Z-DNA and provide a new insight into the effect of DNA bending.

Mapping DNA Conformations Using Single-Molecule Conductance Measurements.

DNA is an attractive material for a range of applications in nanoscience and nanotechnology, and it has recently been demonstrated that the electronic properties of DNA are uniquely sensitive to its sequence and structure, opening new opportunities for the development of electronic DNA biosensors. In this report, we examine the origin of multiple conductance peaks that can occur during single-molecule break-junction (SMBJ)-based conductance measurements on DNA. We demonstrate that these peaks originate from the presence of multiple DNA conformations within the solutions, in particular, double-stranded B-form DNA (dsDNA) and G-quadruplex structures. Using a combination of circular dichroism (CD) spectroscopy, computational approaches, sequence and environmental controls, and single-molecule conductance measurements, we disentangle the conductance information and demonstrate that specific conductance values come from specific conformations of the DNA and that the occurrence of these peaks can be controlled by controlling the local environment. In addition, we demonstrate that conductance measurements are uniquely sensitive to identifying these conformations in solutions and that multiple configurations can be detected in solutions over an extremely large concentration range, opening new possibilities for examining low-probability DNA conformations in solutions.

The role of electrons’ spin in DNA oxidative damage recognition

Formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (OG) is one of the most common forms of DNA oxidative damage found in human cells. Although this damage is prevalent in many disease states, it only marginally influences the structure and stability of double-stranded DNA (dsDNA). Therefore, it is a challenge to establish the mechanism by which this damage is detected by repair enzymes. We investigated the position-dependent effect of the damage on the interactions between dsDNA and oligopeptides using atomic force microscopy. The results were confirmed by monitoring the spin and location-dependent polarizability of the damaged DNA, applying a Hall device. The observations suggest that the interaction of peptide with DNA depends on oxidative damage in the DNA and on its location relative to the point of contact between the peptide and the DNA. Hence, a remote search mechanism for damage in DNA is possible.

DNA Bending Force Facilitates Z-DNA Formation under Physiological Salt Conditions.

Z-DNA, a noncanonical helical structure of double-stranded DNA (dsDNA), plays pivotal roles in various biological processes, including transcription regulation. Mechanical stresses on dsDNA, such as twisting and stretching, help to form Z-DNA. However, the effect of DNA bending, one of the most common dsDNA deformations, on Z-DNA formation is utterly unknown. Here, we show that DNA bending induces the formation of Z-DNA, that is, more Z-DNA is formed as the bending force becomes stronger. We regulated the bending force on dsDNA by using D-shaped DNA nanostructures. The B–Z transition was observed by single-molecule fluorescence resonance energy transfer. We found that as the bending force became stronger, Z-DNA was formed at lower Mg2+ concentrations. When dsDNA contained cytosine methylations, the B–Z transition occurred at 78 mM Mg2+ (midpoint) in the absence of the bending force. However, the B–Z transition occurred at a 28-fold lower Mg2+ concentration (2.8 mM) in the presence of the bending force. Monte Carlo simulation suggested that the B–Z transition stabilizes the bent form via the formation of the B–Z junction with base extrusion, which effectively releases the bending stress on DNA. Our results clearly show that the bending force facilitates the B–Z transition under physiological salt conditions.

Target-induced ratiometric electrochemical aptasensor for highly sensitive detection of thrombin based on AuNPs-MXene

Development of specific and sensitive electrochemical sensing platform for thrombin (TB) analysis is highly desirable for its important roles in many diseases. Herein, we designed and explored a facile ratiometric electrochemical aptasensor for TB detection based on signal on–off strategy. AuNPs-MXene was synthesized, characterized, and used to functionalize the basic electrode as a signal-amplified sensing platform. Hairpin probe (Hp) labeled with ferrocene (Fc) as signal probe, TB aptamer (TBA) labeled with methyl blue (MB) as capture probe are rationally introduced to fabricate highly-selective recognition and ratiometric dual-signal readout aptasensor. The sequence of Fc-Hp was designed to be complementary with MB-TBA to form a double-stranded DNA structure. When TB is present, the specific interaction between MB-TBA and TB leads to the departure of MB-TBA and the formation of hairpin structure of Fc-Hp, resulting in the leaving away of MB and confining Fc group near the electrode surface. Consequently, the oxidation peak current (Ipa) of Fc increased (signal-on), meanwhile Ipa of MB decreased (signal-off). Thus, the specific recognition of MB-TBA to TB can be read out in an electrochemical signal on–off ratiometric mode. A novel ratiometric electrochemical aptasensor was constructed with a wide linear range of 5.0 fmol/L ∼ 1.0 pmol/L and a low detection limit of 1.7 fmol/L (3S0/S). Moreover, this ratiometric electrochemical aptasensor shows excellent selectivity, outstanding sensitivity, and good stability in real serum sample analysis, exhibiting promising potential application in bioanalysis.

A novel and label-free electrochemical aptasensor based on exonuclease III and G-quadruplex DNAzyme for sensitive and selective detection of metronidazole

A novel and label-free electrochemical aptasensor based on exonuclease III (Exo III) and G-quadruplex DNAzyme has been first designed to detect metronidazole (MNZ) sensitively and selectively in this paper. Capture probe (CP), helper probe (HP) and aptamer (Apt) hybridize to form double-stranded DNA (dsDNA) structure with a blunt 3′-terminus, resulting in the guanine (G)-rich CP being enclosed in the dsDNA, followed by specific digestion of the 3′-terminus by Exo III to expose the G-rich sequence. At this time, CP can bind with the added hemin to form G-quadruplex DNAzyme which catalyzes the redox reaction of 3,3′,5,5′-tetramethylbenzidine (TMB) and H2O2, generating a strong current signal. However, in the presence of MNZ, the binding of MNZ and aptamer breaks the original blunt 3′-terminus of the dsDNA structure, blocking the subsequent series of reactions, and only a weak current signal can be observed. Under the optimal conditions, the aptasensor showed excellent sensitivity and specificity for the detection of MNZ with a wide linear range from 10 pM to 500 nM and a detection limit as low as 3.0 pM (S/N = 3). The aptasensor constructed based on MNZ aptamer can effectively avoid the interference of other nitro compounds and antibiotics to a certain extent. Compared with other specific recognition elements, the aptasensor possesses the advantages of strong specificity, simple preparation and low cost. Moreover, the proposed aptasensor has been successfully applied to the detection of MNZ in tap water and honey samples.

STUDY OF STRUCTURAL TRANSITIONS OF COMPLEXES OF DIFFERENT LIGANDS WITH DNA AND BSA

Structural transitions of DNA and bovine serum albumin (BSA) and their complexes with methylene blue (MB) and Hoechst 33258 (H33258) by UV-denaturation method have been studied. The obtained results revel that MB stabilizes both protein dimensional structure and DNA double-stranded structure, while H33258 stabilizes double-stranded structure of DNA and destabilizes protein structure. It was also revealed that the stabilizing effect of MB on native structure of DNA is more pronounced at relatively low ligand concentrations, than on protein.

Development of a graphene oxide nanosheet and double-stranded DNA structure based fluorescent "signal off" aptasensor for ochratoxin A detection in malt.

A "signal off" fluorescent aptasensor based on graphene oxide (GO) nanosheet and double-stranded DNA structure was fabricated for OTA detection. In the absence of OTA, the aptamer and its complementary DNA (cDNA) formed double-stranded conjugates that could coexist with GO, presenting fluorescence responses. Then, the presented OTA was captured by the aptamers, causing the release of cDNA-FAM probes. The free probes were adsorbed by GO, leading to an OTA concentration-dependent fluorescence quenching through fluorescence resonance energy transfer. Under optimum conditions, the fluorescent aptasensor exhibited outstanding sensitivity with a LOD of 11pg/mL and a wide dynamic range of 0.04-30ng/mL. The selectivity of the aptasensor was confirmed against other four mycotoxins, and the feasibility and reliability were verified by determining OTA in the spiked malt samples with satisfactory recovery of 95.50%-112.05%. This aptasensing platform could be adapted to measure other mycotoxins by replacing the aptamer sequences for food safety.

Spectroscopic, electrochemical, and some theoretical studies on the interactional of neuraminidase inhibitor zanamivir with double helix deoxyribonucleic acid

In some cases, instead of designing new molecules, investigating the potential of molecules with small chemical structures with biological activity to interact with double-stranded DNA is very important for the design of drug candidate molecules that can target new DNA. In our literature search, we did not encounter the mode of binding of Zanamivir (ZAN), an antiviral drug, with its double-stranded DNA structure. For this reason, the types of interactions between dsDNA and a ZAN were investigated experimentally and theoretically by comparative experimental and theoretical studies between different groups. This study covers spectroscopic studies such as absorption, fluorescence, and FT-IR, electrochemical properties such as cyclic voltammetry, and some physical properties such as viscosity and theoretical studies at certain pH values. As a result of our theoretical studies (according to the results obtained from the lowest energy insertion poses) supporting our experimental studies, we concluded that ZAN prefers the minor groove binding mechanism and settles into the double helix structure.

Rapid and ultrasensitive electromechanical detection of ions, biomolecules and SARS-CoV-2 RNA in unamplified samples.

The detection of samples at ultralow concentrations (one to ten copies in 100 μl) in biofluids is hampered by the orders-of-magnitude higher amounts of 'background' biomolecules. Here we report a molecular system, immobilized on a liquid-gated graphene field-effect transistor and consisting of an aptamer probe bound to a flexible single-stranded DNA cantilever linked to a self-assembled stiff tetrahedral double-stranded DNA structure, for the rapid and ultrasensitive electromechanical detection (down to one to two copies in 100 μl) of unamplified nucleic acids in biofluids, and also of ions, small molecules and proteins, as we show for Hg2+, adenosine 5'-triphosphate and thrombin. We implemented an electromechanical biosensor for the detection of SARS-CoV-2 into an integrated and portable prototype device, and show that it detected SARS-CoV-2 RNA in less than four minutes in all nasopharyngeal samples from 33 patients with COVID-19 (with cycle threshold values of 24.9-41.3) and in none of the 54 COVID-19-negative controls, without the need for RNA extraction or nucleic acid amplification.