Target DNA Concentration Research Articles

A higher DNA damage rate in aqueous eDNA particles suggests intra‐cellular eDNA degradation in cellular environments

AbstractA higher DNA quality in an environmental DNA (eDNA) sample is crucial for the improved and precise assessment of biodiversity via eDNA analysis. However, the frequency of damage of DNA molecules released from macro‐organisms in aquatic environments remains unclear. As a proof of concept, the rate of DNA damage (λ; per base pair) in an eDNA sample was estimated by measuring the quantity of target DNA with different fragment lengths using quantitative real‐time PCR (qPCR). Rearing water was collected from tanks housing zebrafish (Danio rerio) using filters with different pore sizes ranging from 0.2 to 10 μm, multiple fragment lengths of their mitochondrial eDNA concentrations were quantified and estimated the DNA damage rate based on an exponential relationship between target DNA concentrations and DNA fragment lengths. The DNA damage rate in zebrafish eDNA particles was higher for larger pore size filters, implying that cell membranes do not necessarily protect intra‐cellular DNA from degradation and high activity of intra‐cellular DNases actively degrades intra‐cellular eDNA molecules. Moreover, this finding may partly account for the apparently shorter persistence of larger eDNA particles in aquatic environments and the initial rapid decay of eDNA fragments in a biphasic degradation process. Additional field studies are required to investigate the frequency of DNA damage in different eDNA size fractions and states to facilitate the efficient collection of population genetic information on aquatic macro‐organisms from environmental samples.

An enzyme-free turn-on fluorescent strategy for nucleic acid detection based on hybridization chain reaction and transferable silver nanoclusters.

A fluorescence biosensor has been developed based on hybridisation chain reaction (HCR) amplification coupled with silver nanoclusters (AgNCs) for nucleic acid detection. The fluorescence was activated via end-to-end transfer of dark AgNCs caged within a DNA template to another DNA sequence that could enhance their red fluorescence emission at 611nm. Such cluster-transfer approach allows us to introduce fluorogenic AgNCs as external signal transducers, thereby enabling HCR to perform in a predictable manner. The resulted HCR-AgNC biosensor was able to detect target DNA with a detection limit of 3.35 fM, and distinguish the DNA target from single-base mismatch sequences. Moreover, the bright red fluorescence emission was detectable with the naked eye, with concentration of target DNA down to 1pM. The biosensor also performed well in human serum samples with good recovery. Overall, our cluster-transfer approach provides a good alternative to construct HCR-AgNC assay with less risk of circuit leakage and produce AgNCs in a controllable manner.

Quantification of mRNA in Single Cells Based on Dimerization-Induced Photoluminescence Nonblinking of Quantum Dots.

Photoluminescence (PL) intermittency (or "blinking") is a unique characteristic of single quantum dot (QD) emission. Here, we report a novel single-molecule detection strategy for the intracellular mRNA of interest using the mRNA-induced nonblinking QD dimers as probes. The working principle of the method is that the DNA hybrid of the target DNA (or mRNA) with a biotin-modified ssDNA probe can induce two blinking streptavidin-modified QDs (SAV-QDs) conjugated. The formed QD dimer as a bright spot showed a nonblinking emission property, observed with total inner reflection fluorescence microscopy (TIRFM). In theory, one nonblinking spot indicated a target DNA (or mRNA). The experimental results from single-spot fluorescence trajectory analysis and single-particle brightness analysis based on TIRFM and fluorescence correlation spectroscopy (FCS) techniques verified this dimerization process of QDs or its induced nonblinking emission. Employing a target DNA with the same base sequences to Survivin mRNA as a model, the detection strategy was used to detect the target DNA concentration based on the linear relationship between the percentage of the nonblinking spots and the target DNA concentration. This single-molecule detection strategy was also successfully used for determining Survivin mRNA in a single HeLa cell. The method can simplify the hybridization steps, eliminate self-quenching and photobleaching of fluorophores, and reduce the influence of unspecific binding on the detection.

Dual-enzyme cascade amplification electrochemical biosensor for human papillomavirus based on DNA nanoflower structure

The lack of large-scale human papillomavirus (HPV) DNA screening is a major contributor to the high incidence and mortality of cervical cancer in economically undeveloped areas. The development of sensitive, rapid, and low-cost screening techniques is urgently needed. Here, DNA nanoflowers encapsulating glucose oxidase and horseradish peroxidase (GHDFs) were synthesized by one-pot rolling circle amplification, and then the GHDFs were used as the cargo of DNA hydrogel and applied for HPV DNA detection. When target DNA was present, the DNA hydrogel cross-linking structure was disrupted, releasing GHDFs, which then catalysed the oxidation of glucose and tetramethylbenzidine in a cascade, generating a significant electrical signal. Signal intensity had a linear relationship with the logarithm of target DNA concentration in the range of 10 fM-1 nM with a detection limit of 3.76 fM. The detection time of the proposed biosensor was 25 min, which was suitable for large-scale HPV DNA screening in economically underdeveloped areas and provides a blueprint for the detection of other DNA of interest.

Electrochemical DNA Biosensor Based on Mercaptopropionic Acid-Capped ZnS Quantum Dots for Determination of the Gender of Arowana Fish

A new electrochemical DNA biosensor based on mercaptopropionic acid (MPA)-capped ZnS quantum dots (MPA-ZnS QDs) immobilization matrix for covalent binding with 20-base aminated oligonucleotide has been successfully developed. Prior to the modification, screen-printed carbon paste electrode (SPE) was self-assembled with multilayer gold nanoparticles (AuNPs) and cysteamine (Cys). The inclusion of MPA-ZnS QDs semiconducting material in modified electrodes has enhanced the electron transfer between the SPE transducer and DNA leading to improved bioanalytical assay of target biomolecules. Electrochemical studies performed by cyclic voltammetry (CV) and differential pulsed voltammetry (DPV) demonstrated that the MPA-ZnS QDs modified AuNPs electrode was able to produce a lower charge transfer resistance response and hence higher electrical current response. Under optimal conditions, the immobilized synthetic DNA probe exhibited high selectivity towards synthetic target DNA. Based on the DPV response of the reduction of anthraquinone monosulphonic acid (AQMS) redox probe, the MPA-ZnS QDs-based electrochemical DNA biosensor responded to target DNA concentration from 1 × 10−9 μM to 1 × 10−3 μM with a sensitivity 1.2884 ± 0.12 µA, linear correlation coefficient (R2) of 0.9848 and limit of detection (LOD) of 1 × 10−11 μM target DNA. The DNA biosensor exhibited satisfactory reproducibility with an average relative standard deviation (RSD) of 7.4%. The proposed electrochemical transducer substrate has been employed to immobilize the aminated Arowana fish (Scleropages formosus) DNA probe. The DNA biosensor showed linearity to target DNA from 1 × 10−11 to 1 × 10−6 µM (R2 = 0.9785) with sensitivity 1.1251 ± 0.243 µA and LOD of 1 × 10−11 µM. The biosensor has been successfully used to determine the gender of Arowana fish without incorporating toxic raw materials previously employed in the hazardous processing conditions of polypyrrole chemical conducting polymer, whereby the cleaning step becomes difficult with thicker films due to high levels of toxic residues from the decrease in polymerization efficacy as films grew.

Design of Species-Specific Primers for Early Detection of Kretzschmaria zonata, the Causal Agent of Root and Neck Rot of Teak (Tectona grandis)

Kretzschmaria zonata (Lév.) P.M.D. Martin is a fungus that causes root and neck rot in teak (Tectona grandis L.) worldwide. The detection of this fungus in asymptomatic plants is essential for its control but, to date, the disease can only be detected morphologically or by sequencing this fungus’s isolates. The aim of this work was to design, at least, one set of specific PCR primers for a quickly, early and accurate diagnosis of this pathogen. Nineteen isolates of K. zonata from teak plants at different locations in Mexico were obtained. ITS region sequences and two software were used to design the PCR-based primers. All primers obtained were tested on DNA samples from infected teak tissue, K. zonata pure isolates, and other different fungi species (in vitro and in silico). The sensitivity of the primers was evaluated at five different concentrations of target DNA. The primer set KZ-AQ-3F/KZ-AQ-3R was selected for its specificity (amplifies only K. zonata) and sensitivity (1 × 10−5 ng/µL detection). This is the first report of a Kretzschmaria zonata-specific primer set. Tests carried out on asymptomatic teak and artificially inoculated plants with this fungus demonstrated the usefulness of these primers for the accurate detection of K. zonata, and taking early control measures.

Bipolar Electrode Arrays for Chemical Imaging and Multiplexed Sensing.

Bipolar electrodes (BPEs) with arrays of cathodic and anodic poles were developed for use in closed bipolar systems. To increase the number of BPEs in the array, the anodic and cathodic poles were connected with each other using thin leads. A further increase in the number of BPEs was achieved by forming the cathodic and anodic poles of the BPEs and the leads in different layers. A device with 9 × 10 arrays of cathodes and anodes was thus realized. When using this device to sense hydrogen peroxide (H2O2), the sensitivity and linear range of calibration plots could be adjusted by changing the driving voltage and the area ratio between the cathodic and anodic poles. The devices were used to image H2O2 and obtain time-lapse images for the diffusion and dilution of H2O2. Furthermore, DNA detection was demonstrated using an electroactive intercalator. The sensitivity could be improved by making the anodic poles smaller with respect to the cathodic pole and concentrating the electrochemiluminescence (ECL) in a small area. The ECL intensity changed according to the target DNA concentration in the solution.

Enhanced chemiluminescence imaging sensor for ultrasensitive detection of nucleic acids based on HCR-CRISPR/Cas12a

CRISPR/Cas systems have ignited increasing attention in accurate and sensitive nucleic acids detection. In this work, we proposed the first CRISPR/Cas12a-based chemiluminescence enhancement biosensor by employing HCR amplifying strategy (CLE-CRISPR) for nucleic acids detection, which shows the advantages of high sensitivity and specificity, low-cost, visual imaging by comparison to reported biosensors. Upon the DNA target recognition, the activated CRISPR/Cas12a enabled randomly cutting initiator DNA (intDNA) into vast short products, which could not trigger the toehold-mediated DNA-strand displacement reaction (TSDR) with MB@crDNA. Thereby, the terminus of crDNA induced the hybridization chain reaction (HCR) with the coexistence of two hairpins (H1 and H2), forming a long double-stranded DNA framework. The attached streptavidin-AP yielded a conspicuous CL signal or visual imaging directly related to the DNA target concentration. The proposed CLE-CRISPR platform exhibited excellent sensitivity, with a relatively low detection limit at 3 pM for synthetic DNA target and single copy detection for plasmid by combining recombinase polymerase amplification (RPA) kit. We further validated the practical application of this platform using HPV clinical samples, achieving superior sensitivity and specificity of 88.89% and 100%, respectively. We believe that this work not only extends the application scope of CRISPR/Cas12a, but also devotes a new approach for clinical diagnosis.

A zero-background electrochemical DNA sensor coupling ligase chain reaction with lambda exonuclease digestion for CYP2C19*2 allele genotyping in clinical samples

A zero-background electrochemical DNA sensor is badly desired for highly accurate real-sample assay. Herein, we built a lambda exonuclease (λ-Exo)-assisted ligase chain reaction (LCR)-based electrochemical biosensor, named λ-eLCR, for highly sensitive CYP2C19*2 allele genotyping with zero-background by using the methylene blue (MB)-attached DNA probe and an inverted hybridization format. Specifically, the ligated MB-labeled single-stranded DNAs (ssDNA) were generated exponentially by LCR amplification followed by λ-Exo digestion in the presence of target DNA, then they were invertedly hybridized with the upper sequence of capture probes on the gold electrode, bringing the redox reporter-MB close enough to the surface of the electrode. In this regard, the MB-derived signal detected by square wave voltammetry technique was quantitatively related with the target DNA concentration and guaranteed the zero background. Under the optimum condition, the proposed λ-eLCR exhibited excellent performance in terms of good linear range (100 fM–10 pM) and a low limit of detection (100 fM) for CYP2C19*2 gene. Finally, this λ-eLCR successfully distinguished the CYP2C19*2 allele genotypes in human whole blood samples, which has a great prospect in individualized medicine.

Programmable graphene-based microfluidic sensor for DNA detection

This study presents the development of a lab-on-a-chip (LoC) by integrating a graphene field-effect transistor (FET) chip with a programmable microfluidic device for DNA detection. The real-time biochemical events on the graphene FET chip were monitored through Dirac voltage shift data from the portable graphene curve reader with changes dependent on the fluidic flow into the sensing interface by a fully automated programmable microfluidic system. High sensitivity with high reliability can be obtained with a nine-graphene sensor layout on a single chip. The portable graphene curve reader also provides a tunable electrical parameter setup and straightforward data acquisition. Fluidic control was performed through a multi-position valve, allowing sequential commands for liquid injection into the polydimethylsiloxane (PDMS) flow cell mounted on the sensing chip. The flow cell design with impinging jet geometry and the microfluidic system packaging offer high precision and portability as a less laborious and low-cost sensing setup. The merged system allows for various functionalities, including probe DNA (pDNA) immobilization, a blocking step, and DNA hybridization with stable signal output autonomously, even in a long-run experimental setup. As a DNA sensor, the proposed prototype has demonstrated a high sensitivity of ~44 mV/decade of target DNA concentration, with an outstanding limit of detection (LoD) of ~0.642 aM, making it one of the most sensitive sensors reported up to date. The programmable device has demonstrated essential versatilities for biomolecular detection in a fully portable and automated platform.

A label-free electrochemical impedimetric DNA biosensor for genetically modified soybean detection based on gold carbon dots.

A label-free electrochemical impedimetric biosensor was constructed based on gold carbon dots (GCDs) modified screen-printed carbon electrode for the detection of genetic modified (GM) soybean. The structure and property of GCDs were investigated. The GCDs can directly bind to single-stranded DNA probes through Au-thiol interaction and boost electric conductivity for the DNA sensor construction. The quantification of target DNA was monitored by the change of electron-transfer resistance (Ret) upon the DNA hybridization on sensor surface. Under the optimal conditions, the Ret response (vs. Ag reference electrode) increased with the logarithm of target DNA concentrations in a wide linear range of 1.0 × 10-7 - 1.0 × 10-13M with a detection limit of 3.1 × 10-14M (S/N = 3). It was also demonstrated that the proposed DNA sensor possessed high specificity for discriminating target DNA from mismatched sequences. Moreover, the developed biosensor was applied to detect SHZD32-1 in actual samples, and the results showed a good consistency with those obtained from the gel electrophoresis method. Compared with the previous reports for DNA detection, the label-free biosensor showed a comparatively simple platform due to elimination of complicated DNA labeling. Therefore, the proposed method showed great potential to be an alternative device for simple, sensitive, specific, and portable DNA sensor.

Electrochemiluminescence biosensor for HPV16 detection based on the adjusting of steric hindrance effect coupled with Exonuclease III amplification strategy

The effect of steric hindrance of electrode surface can affect the diffusion of the Ru(bpy)32+ toward electrode and thus in turn affect the ECL performance of the system. In this study, this character had been adopted to develop a biosensor for HPV DNA detection. Exonuclease III (Exo III) signal amplification strategy had been applied to realize signal amplification. First, hairpin probes (HP) was anchored on the surface of electrode as capture probes, HP can resistant to the hydrolyzation of Exo III due to its 3′-protruding termini. Without the target, a large amount of cDNA modified Au nanocages (AuNCs-cDNA) can hybridize with HP and connected to surface of electrode, weak ECL signals can be detected since Ru(bpy)32+ can not diffuse freely to the electrode surface because of the steric hindrance of AuNCs-cDNA. In the presence of the target, HP can hybridize with the target to form double-stranded DNA (dsDNA) with a blunt 3′ terminus, due to the high preference of Exo III for cleaving dsDNA with a blunt 3′ termini, HP in dsDNA was hydrolyzed, and the target which formed dsDNA was released to hybridize with another HP, inducing the Exo III assisted amplification strategy. Due to the reduction of HP on electrode surface, the amount of AuNCs-cDNA connected to the electrode surface become small, a high ECL signal can be detected. Under the optimal conditions, the ECL response of the system has a linear relationship with logarithm of target DNA concentration in the range of 10 fM to 100 pM, and a detection limit of 3.54 fM (S/N = 3). The proposed biosensor has high sensitivity and selectivity, which had been applied to the detection of target DNA in real sample and the satisfied results had been obtained. This system also can detect different targets by changing the DNA sequence easily.

Organometallic hotspot engineering for ultrasensitive EC-SERS detection of pathogenic bacteria-derived DNAs

The sensitivity and limit-of-detection (LOD) of the traditional surface-enhanced Raman spectroscopy (SERS) platform suffer from the requirement of precise positioning of small analytes, including DNAs and bacteria, into narrow hotspots. In this study, a novel SERS sensor was developed using electrochemical deposition onto metal nanopillars (ECOMPs) combined with complementary DNAs (cDNAs) for the detection of pathogenic bacteria. Applying a redox potential to AuCl4− ions actively engineered the organometallic hotspots based on the cDNAs in a short time (<10 min) and simultaneously produced SERS signals. Because of the influence of potential-driven morphological properties on the SERS efficiency in the cDNA domains and the resonant coupling of internal fields with the fields confined between adjacent ECOMPs-cDNAs, the optimum growth time was determined to be 5 min. The EC-SERS detection and discrimination of Enterococcus faecium and Staphylococcus aureus were successfully carried out because of the DNA complementarity. Compared with plasmonic metal nanopillars (MPs)-cDNAs, the enhancement factor of the ECOMPs-cDNAs was estimated to be ∼2.0 × 103. A quantitative investigation revealed that a highly linear progression in the target DNA concentration range (0.05–100 nM) and a LOD of ∼0.035 nM were achieved. The specificity of the ECOMPs-cDNAs was validated by cross-hybridization. The platform was also used to assay human whole blood containing 0.1 nM bacterial DNAs. The proposed strategy provides the potential for highly sensitive SERS-based multiplex DNA detection in clinical diagnostics.

Development of a real-time PCR assay to detect and quantify the tropical root-knot nematode (Meloidogyne arenaria) in latently infected potato seed

Tropical root-knot nematodes (M. incognita, M. arenaria and M. javanica) are a serious problem to most crops, both cultivated and non-cultivated. In the recent past, accurate detection and discrimination of tropical Meloidogyne spp. has been achieved through the development of DNA-based diagnostic assays among others real-time polymerase chain reaction (qPCR) to complement results obtained using morphological and biochemical-based diagnostic methods. However, despite the developments, most of the current methods have not been optimized to quantify and characterize the tropical Meloidogyne spp. particularly in latent infections. Currently, this is a big problem in potato seed production where latently infected tubers often get distributed resulting in the distribution of root-knot nematodes to new areas. In this study, we sought to develop and validate a diagnostic method of quantifying root-knot nematode, M. arenaria in latently infected potato seed tubers. Study findings indicated that there is a high (R2 &gt; 0.953) and significant (P &lt; 0.05) positive correlation between target DNA concentration and Ct values, and the assays can be used to quantify as low as 1.53/100th of DNA associated with individual juvenile nematodes. Using high resolution melting curve analysis, Meloidogyne arenaria samples produced specific melting peaks (79.32 ± 0.029°C, P &lt; 0.05) clearly distinguishing themselves from other tropical Meloidogyne species (M. incognita 79.50 ± 0.022°C and M. javanica 79.96 ± 0.046°C). The development of the high-resolution melting curve (HRMC) analysis method for quantification and characterization of M. arenaria in this study will greatly improve on accurate screening of this pathogen during latent infections to curb potato losses often associated with distribution of infected seed. Key words: Root-knot nematode, real-time PCR, high resolution melting curve analysis, South Africa, Meloidogyne spp. and potato seed

Electrochemical DNA Biosensors Based on the Intrinsic Topological Insulator BiSbTeSe2 for Potential Application in HIV Determination.

In this work, we reported a sensitive, label-free electrochemical biosensor based on the intrinsic topological insulator (TI) BiSbTeSe2 for potential application in the determination of the HIV gene. With strong spin-obit coupling, TIs could have robust surface states with low electronic noise, which might be beneficial for the stable and sensitive electron transport between the electrode and electrolyte interface. Under optimized conditions of the biosensors using BiSbTeSe2, the differential pulse voltammetry (DPV) peak currents showed a linear relationship with the logarithm of target DNA concentrations ranging from 1.0 × 10-13 to 1.0 × 10-7 M, with a detection limit of 1.07 × 10-15 M. The sensing assay also displayed good selectivity and stability after storage at 4 °C for 7 days. This work provides an effective way to develop biosensors with topological materials, which have a potential application in the clinical determination and monitoring field.

DNA hybridization-induced fluorescence variation in ThT: a new strategy of developing aqueous sensors for MO genes.

Mycoplasma ovipneumoniae (MO), a disease-causing pathogen with some of the highest levels of morbidity and mortality, can spread silently at the herd level. A novel alternative nanoprobe for MO was prepared using porous metal-organic frameworks (MOFs) as the scaffold and hairpin DNA with specific sequences of MO and G4 segments as the probe. This preparation was based on the strong fluorescence emission by ThT (thioflavin T) in the limitation of G-quadruplexes with a cavity structure. The use of MOFs effectively limited the folding behavior of G4 as a part of the probe to improve the defect of the strong background signal caused by the free-state G4Probe in a buffer. The results from the selectivity experiment showed that only a trace amount of the target with lower ΔG could be the "key" to the highly efficient triggering of the release behavior of the G4Probe from MOFs and the subsequent change in the fluorescence behavior of ThT. The DNA targets could be determined by observing the change in the signal. More importantly, the probe showed a low detection limit and a good linear correlation between the concentration of target DNA ranging from 10-10 M to 10-6 M not only in buffer but also in natural complex media. Moreover, the operation involved in the whole strategy was simple and the total cost was low. These findings demonstrated the value of the probe in further clinical diagnosis. This study reports the successful construction of a ΔG-sensitive sensor for MO for the first time.

Electrochemical Resistive DNA Biosensor for the Detection of HPV Type 16

In this work, a low-cost and rapid electrochemical resistive DNA biosensor based on the current relaxation method is described. A DNA probe, complementary to the specific human papillomavirus type 16 (HPV-16) sequence, was immobilized onto a screen-printed gold electrode. DNA hybridization was detected by applying a potential step of 30 mV to the system, composed of an external capacitor and the modified electrode DNA/gold, for 750 µs and then relaxed back to the OCP, at which point the voltage and current discharging curves are registered for 25 ms. From the discharging curves, the potential and current relaxation were evaluated, and by using Ohm’s law, the charge transfer resistance through the DNA-modified electrode was calculated. The presence of a complementary sequence was detected by the change in resistance when the ssDNA is transformed in dsDNA due to the hybridization event. The target DNA concentration was detected in the range of 5 to 20 nM. The results showed a good fit to the regression equation ∆Rtotal (Ω) = 2.99 × [DNA] + 81.55, and a detection limit of 2.39 nM was obtained. As the sensing approach uses a direct current, the electronic architecture of the biosensor is simple and allows for the separation of faradic and nonfaradaic contributions. The simple electrochemical resistive biosensor reported here is a good candidate for the point-of-care diagnosis of HPV at a low cost and in a short detection time.

Capability of novel fluorescence DNA-conjugated CdTe/ZnS quantum dots nanoprobe for COVID-19 sensing

Urgent identification of COVID-19 in infected patients is highly important nowadays. Förster or fluorescence resonance energy transfer (FRET) is a powerful and sensitive method for nanosensing applications, and quantum dots are essential materials in FRET-based nanosensors. The QDs are conjugated to DNA or RNA and used in many applications. Therefore, in the present study, novel fluorescence DNA-conjugated CdTe/ZnS quantum dots nanoprobe designed for detection of Covid-19 after extracting their RNA from saliva of hesitant people. For achieving this purpose, the water-soluble CdTe/ZnS QDs-DNA prepared via replacing the thioglycolic acid (TGA) on the surface of QDs with capture DNA (thiolated DNA) throw a ligand-exchange method. Subsequently, by adding the different concentrations of complementary (target DNA) in a mixture of quencher DNA (BHQ2-labeled DNA) and the QDs-DNA conjugates at different conditions, sandwiched hybrids were formed. The results showed that the fluorescence intensity was decreased with increasing the concentration of target DNA (as a positive control). The linear equation and regression (Y = 40.302 X + 1 and R2 = 0.98) were obtained by using the Stern-Volmer relationship. The Limit of detection (LOD) was determined 0.000823 µM. The achieved results well confirm the outcomes of the RT-PCR method in real samples.

1052. Characterisation of the DNA binding properties of ridinilazole, a selective antibiotic currently in phase III trials for the treatment of Clostridioides difficile

Background Clostridioides difficile infection (CDI) is recognised by the CDC as an “urgent threat” in the USA, responsible for nearly 13,000 deaths, and carries an economic burden ranging from &5.4 to &6.3 billion per year. In a phase II study, ridinilazole was shown to be effective at treating CDI and decreasing subsequent recurrence compared to vancomycin. However, the precise mechanism of action of ridinilazole has yet to be fully elucidated. We now present data that reveals ridinilazole clearly co-localises with DNA in C. difficile and binds with high affinity to the minor groove of DNA. These interactions are predicted to have consequences on cellular functions within C. difficile.MethodsHigh resolution confocal microscopy was used to track the intracellular localisation of ridinilazole in C. difficile. Fluorescence intensity was used to characterise the DNA binding properties of ridinilazole; sequence specificity was demonstrated with AT- or GC-rich DNA polymers, and tight binding was shown using short double-stranded oligonucleotides. Hanging drop vapour diffusion enabled co-crystallisation and subsequent structural determination of DNA-bound ridinilazole. ResultsConfocal microscopy revealed clear co-localisation of ridinilazole to the DNA within C. difficile. Ridinilazole demonstrated a dose-dependent increase in fluorescence in response to increasing concentration of target DNA. Fluorescence binding studies revealed that ridinilazole shows a preference towards AT-rich DNA sequences. Tight binding characteristics were demonstrated by ridinilazole in complex with short double-stranded oligonucleotides, returning dissociation constants (Kd) of 20 – 50 nM. Crystallisation enabled co-structures of ridinilazole bound to the minor groove of double-stranded DNA oligonucleotides to be solved.ConclusionRidinilazole demonstrates tight binding with sequence specificity within the minor groove of DNA and co-localises with DNA in C. difficle. Further analysis is ongoing to fully understand this novel mechanism of action, the downstream consequences of these interactions and how they contribute to the bactericidal activity of ridinilazole.Disclosures Clive Mason, PhD, Summit Therapeutics (Employee, Shareholder) Tim Avis, n/a, Summit therapeutics (Shareholder) Chris Coward, PhD, Summit Therapeutics (Employee, Scientific Research Study Investigator, Shareholder) David Powell, PhD, Summit Therapeutics (Employee) Kevin W. Garey, Pharm.D., M.S., FASHP, Summit Therapeutics (Research Grant or Support)

SERS-PCR assays of SARS-CoV-2 target genes using Au nanoparticles-internalized Au nanodimple substrates

The reverse transcription-polymerase chain reaction (RT-PCR) method has been adopted worldwide to diagnose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although this method has good sensitivity and specificity, there is a need to develop a more rapid diagnostic technology, given the virus’s rapid spread. However, the RT-PCR method takes a long time to diagnose SARS-CoV-2 because of the required thermocycling steps. Therefore, we developed a surface-enhanced Raman scattering (SERS)-PCR detection method using an AuNP-internalized Au nanodimple substrate (AuNDS) to shorten the diagnosis time by reducing the number of thermocycling steps needed to amplify the DNA. For the representative target markers, namely, the envelope protein (E) and RNA-dependent RNA polymerase (RdRp) genes of SARS-CoV-2, 25 RT-PCR thermocycles are required to reach a detectable threshold value, while 15 cycles are needed for magnetic bead-based SERS-PCR when the initial DNA concentration was 1.00× 105 copies/μL. However, only 8 cycles are needed for the AuNDS-based SERS-PCR. The corresponding detectable target DNA concentrations were 3.36 × 1012, 3.28 × 109, and 2.56 × 107 copies/μL, respectively. Therefore, AuNDS-based SERS-PCR is seen as being a new molecular diagnostic platform that can shorten the time required for the thermocycling steps relative to the conventional RT-PCR.