Detection Of Nucleic Acid Sequences Research Articles

Modern trends in the application of thermoelectric method in non-destructive testing (review)

The article provides an overview of the main directions of using the thermoelectric testing method in various spheres of the national economy. The thermoelectric method is most widely used in industry. There are publications on the application of the thermoelectric method for quality monitoring of turning and friction stir welding. It is shown that the thermoelectric method makes it possible to increase the reliability of testing and, based on testing data, ensure optimal modes of the technological process of metalworking and welding. A number of articles are devoted to the application of the thermoelectric method to plastic deformation testing, the dependence of thermal EMF on the degree of plastic deformation is revealed. Recently, publications have appeared on the application of the thermoelectric method to testing the thermal resistance of the design “housing of a power semiconductor device-thermal interface-cooling radiator”. This design is very widespread in electronic equipment. In addition, there are articles on the use of the thermoelectric method to testing the transient resistance of contacts in the power supply network. It is shown that with an increase in contact resistance, the value of thermal EMF increases proportionally, which can be used to prevent emergencies in the power supply network. The thermoelectric method has also been successfully applied to diagnose the degree of titanium flooding. The dependence of the thermal EMF value on the degree of hydrogenation has been revealed. The use of the thermoelectric method is not limited to the field of industrial production. The thermoelectric method is successfully used in medicine for the undetectable detection of nucleic acid sequences, for the temperature diagnosis of human teeth, as well as for the diagnosis of inflammatory processes in the human body.

Design and Evaluation of a Robust CRISPR Kinetic Assay for Hot-Spot Genotyping.

Next-generation sequencing offers highly multiplexed and accurate detection of nucleic acid sequences but at the expense of complex workflows and high input requirements. The ease of use of CRISPR-Cas12 assays is attractive and may enable highly accurate detection of sequences implicated in, for example, cancer pathogenic variants. CRISPR assays often employ end-point measurements of Cas12 trans-cleavage activity after Cas12 activation by the target; however, end point-based methods can be limited in accuracy and robustness by arbitrary experimental choices. To overcome such limitations, we develop and demonstrate here an accurate assay targeting a mutation of the epidermal growth factor gene implicated in lung cancer (exon 19 deletion). The assay is based on characterizing the kinetics of Cas12 trans-cleavage to discriminate the mutant from wild-type targets. We performed extensive experiments (780 reactions) to calibrate key assay design parameters, including the guide RNA sequence, reporter sequence, reporter concentration, enzyme concentration, and DNA target type. Interestingly, we observed a competitive reaction between the target and reporter molecules that has important consequences for the design of CRISPR assays, which use preamplification to improve sensitivity. Finally, we demonstrate the assay on 18 tumor-extracted amplicons and 100 training iterations with 99% accuracy and discuss discrimination parameters and models to improve wild type versus mutant classification.

Application of CRISPR/Cas12a for SARS-CoV-2 Nucleic Acid Detection

SARS-CoV-2 spans 3 years, and although the virus has been adjusted from "Category B, Tube A" to "Category B, Tube B" it is still present in the environment, causing widespread epidemic infections and great impact on human health and the environment. Currently, there are various nucleic acid detection methods for this virus, and CRISPR methods are widely used to construct biosensors for accurate and rapid specific detection of nucleic acid sequences due to their powerful gene-editing capabilities. In this article, we introduce the application of different forms of sensors such as fluorescent paper strips built with CRISPR/Cas12a for SARS-CoV-2 nucleic acid detection.

Polydopamine functionalized dendritic fibrous silica nanoparticles as a generic platform for nucleic acid-based biosensing

Accurate and rapid detection of nucleic acid sequences is of utmost importance in various fields, including disease monitoring, clinical treatment, gene analysis and drug discovery. In this study, we developed a "turn-on" fluorescence biosensor that enables simple and highly efficient detection of nucleic acid biomarkers. Our approach involves the utilization of 6-carboxyfluorescein modified single-stranded DNA (FAM-ssDNA) as molecular recognition element, along with polydopamine-functionalized dendritic fibrous nanosilica (DFNS). FAM-ssDNA serves as both specific molecular recognition element for the target analyte and reporter capable of transducing a detectable signal through Watson–Crick base pairing. The polydopamine-functionalized DFNS (DFNS@DA) exhibits strong binding to FAM-ssDNA via polyvalent metal mediated coordination leading to effective quenching by fluorescence resonance energy transfer. In the presence of a complementary target sequence, FAM-ssDNA forms hybridized structure and detaches from DFNS@DA, which causes an increased fluorescence emission. The analytical system based on FAM-ssDNA and DFNS@DA demonstrates exceptional sensitivity, selectivity, and rapid response for the detection of nucleic acid sequences, leveraging the high adsorption and quenching properties of DFNS@DA. For the first proof of concept, we demonstrated the successful detection of microRNA (miR-21) in cancer cells using the FAM-ssDNA/DFNS@DA system. Our results highlight the promising capabilities of DFNS@DA and nucleic acid-based biosensors, offering a generic and cost-effective solution for the detection of nucleic acid-related biomarkers.Graphical abstract

SABER-FISH in Hydractinia.

In situ hybridization allows the detection of nucleic acid sequences in fixed cells and tissues. The gelatinous nature of cnidarians and Hydractinia demands extensive and exhausting protocols to detect RNA transcripts with traditional methods (e.g., colorimetric in situ hybridization). Signal amplification by exchange reaction (SABER) fluorescence in situ hybridization (FISH) enables simplifying and multiplex imaging of RNA targets in a rapid and cost-effective manner. In one enzymatic reaction, SABER-FISH uses a strand-displacing polymerase and catalytic DNA hairpin to generate FISH probes with adjustable signal amplification, allowing highly sensitive detection of nucleic acids and reducing the number of required probes. Here I describe the methodology to detect transcripts within the cells of Hydractinia by SABER-FISH in whole-mount samples.

Rapid detection of nucleic acid sequences of pathogenic bacteria based on a series piezoelectric quartz crystal sensor with transcription activator-like effectors

Accurate and rapid detection of pathogenic bacteria is of great importance in the field of clinical diagnosis and food safety. Current methods for pathogenic bacteria detection have some problems in accurate, rapid and universal application. Here we proposed a pathogenic bacteria series piezoelectric quartz crystal (SPQC) sensor for achieving highly specific and sensitive detection of pathogenic bacteria. The universal sequences of common clinical pathogens screened by our group were used as detection targets. A new TALEs nuclease was synthesized as a recognition element, which recognizes double-stranded DNA at the level of a single base mismatch in the range of 17–19 bases. Targets could be specifically recognized by TALEs, resulting in the change of electrode surface, which would be further amplified by hybridization chain reaction and silver staining technique. Finally, the changes would be detected by SPQC system. This strategy was demonstrated to have excellent performance, enabling sensitive detection of targets with a detection limit of 25 cfu/mL in less than 3 h. What's more, the identification of single base mismatch could be achieved when the target ranging in length between 17 and 19 bases. The proposed method is rapid, accurate and easy universal application and expected to be applied in clinical diagnosis and food safety.

A mutation-sensitive, multiplexed and amplification-free detection of nucleic acids by stretching single-molecule tandem hairpin probes.

The detection of nucleic acid sequences in parallel with the discrimination of single nucleotide variations (SNVs) is critical for research and clinical applications. A few limitations make the detection technically challenging, such as too small variation in probe-hybridization energy caused by SNVs, the non-specific amplification of false nucleic acid fragments and the few options of dyes limited by spectral overlaps. To circumvent these limitations, we developed a single-molecule nucleic acid detection assay without amplification or fluorescence termed THREF (hybridization-induced tandem DNA hairpin refolding failure) based on multiplexed magnetic tweezers. THREF can detect DNA and RNA sequences at femtomolar concentrations within 30 min, monitor multiple probes in parallel, quantify the expression level of miR-122 in patient tissues, discriminate SNVs including the hard-to-detect G-U or T-G wobble mutations and reuse the probes to save the cost. In our demonstrative detections using mock clinic samples, we profiled the let-7 family microRNAs in serum and genotyped SARS-CoV-2 strains in saliva. Overall, the THREF assay can discriminate SNVs with the advantages of high sensitivity, ultra-specificity, multiplexing, reusability, sample hands-free and robustness.

Ultrasensitive and Specific Clustered Regularly Interspaced Short Palindromic Repeats Empowered a Plasmonic Fiber Tip System for Amplification-Free Monkeypox Virus Detection and Genotyping.

The urgent necessity for highly sensitive diagnostic tools has been accentuated by the ongoing mpox (monkeypox) virus pandemic due to the complexity in identifying asymptomatic and presymptomatic carriers. Traditional polymerase chain reaction-based tests, despite their effectiveness, are hampered by limited specificity, expensive and bulky equipment, labor-intensive operations, and time-consuming procedures. In this study, we present a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-based diagnostic platform with a surface plasmon resonance-based fiber tip (CRISPR-SPR-FT) biosensor. The compact CRISPR-SPR-FT biosensor, with a 125 μm diameter, offers high stability and portability, enabling exceptional specificity for mpox diagnosis and precise identification of samples with a fatal mutation site (L108F) in the F8L gene. The CRISPR-SPR-FT system can analyze viral double-stranded DNA from mpox virus without amplification in under 1.5 h with a limit of detection below 5 aM in plasmids and about 59.5 copies/μL when in pseudovirus-spiked blood samples. Our CRISPR-SPR-FT biosensor thus offers fast, sensitive, portable, and accurate target nucleic acid sequence detection.

XNAs: A Troubleshooter for Nucleic Acid Sensing.

The strategies for nucleic acid sensing based on nucleic acid hybridization between the target sequence and the capture probe sequence are considered to be largely successful as far as detection of a specific target of known sequence is concerned. However, when compared with other complementary methods, like direct sequencing, a number of results are still found to be either “false positives” or “false negatives”. This suggests that modifications in these strategies are necessary to make them more accurate. In this minireview, we propose that one way toward improvement could be replacement of the DNA capture probes with the xeno nucleic acid or XNA capture probes. This is because the XNAs, especially the locked nucleic acid, the peptide nucleic acid, and the morpholino, have shown better single nucleobase mismatch discrimination capacity than the DNA capture probes, indicating their capacity for more precise detection of nucleic acid sequences, which is beneficial for detection of gene stretches having point mutations. Keeping the current trend in mind, this minireview will include the recent developments in nanoscale, fluorescent label-free applications, and present the cases where the XNA probes show clear advantages over the DNA probes.

Oxygen vacancy modulated MnO2 bi-electrode system for attomole-level pathogen nucleic acid sequence detection

Electrochemical nucleic acid detection has recently opened a relatively low budget, straightforward and speedy approach of detection with the aid of an electrochemically active redox probe. However, electrochemical sensors of nucleic acid amplification characteristically employ a tri-electrode geometry involving standard reference electrode usually made up of a noble metal that adds up the cost of detection. This work proposes a sensitive and rapid approach for detecting the endpoint of nucleic acid amplification test using a novel bi-electrode sensing geometry. The sensing layer of the electrode was comprised of a transition metal oxide to tune its electronic state for regulating its electrochemical response. The XPS analysis indicates a higher contribution of Mn3+ oxidation state than Mn4+ when annealed at elevated temperature, suggesting the formation of oxygen vacancies into the sensing layer. The formation of oxygen vacancies improved the electrical conduction of the layer as evident from the significant reduction in charge transfer resistance (Rct) value from electrochemical impedance spectroscopy (EIS) measurements. The fabricated device was then used to detect dengue virus sequence DNA (using rolling circle amplification) and Staphylococcus aureus genomic DNA (using polymerase chain reaction) with the limit of detection in the order of 103 target DNA copies

Optimizing locked nucleic acid modification in double-stranded biosensors for live single cell analysis.

Double-stranded (ds) biosensors are homogeneous oligonucleotide probes for detection of nucleic acid sequences in biochemical assays and live cell imaging. Locked nucleic acid (LNA) modification can be incorporated in the biosensors to enhance the binding affinity, specificity, and resistance to nuclease degradation. However, LNA monomers in the quencher sequence can also prevent the target-fluorophore probe binding, which reduces the signal of the dsLNA biosensor. This study investigates the influence of LNA modification on dsLNA biosensors by altering the position and amount of LNA monomers present in the quencher sequence. We characterize the fluorophore-quencher interaction, target detection, and specificity of the biosensor in free solution and evaluate the performance of the dsLNA biosensor in 2D monolayers and 3D spheroids. The data indicate that a large amount of LNA monomers in the quencher sequence can enhance the specificity of the biosensor, but prevents effective target binding. Together, our results provide guidelines for improving the performance of dsLNA biosensors in nucleic acid detection and gene expression analysis in live cells.

Kinetic Analysis of the Interaction of Nicking Endonuclease BspD6I with DNA.

Nicking endonucleases (NEs) are enzymes that incise only one strand of the duplex to produce a DNA molecule that is ‘nicked’ rather than cleaved in two. Since these precision tools are used in genetic engineering and genome editing, information about their mechanism of action at all stages of DNA recognition and phosphodiester bond hydrolysis is essential. For the first time, fast kinetics of the Nt.BspD6I interaction with DNA were studied by the stopped-flow technique, and changes of optical characteristics were registered for the enzyme or DNA molecules. The role of divalent metal cations was estimated at all steps of Nt.BspD6I–DNA complex formation. It was demonstrated that divalent metal ions are not required for the formation of a non-specific complex of the protein with DNA. Nt.BspD6I bound five-fold more efficiently to its recognition site in DNA than to a random DNA. DNA bending was confirmed during the specific binding of Nt.BspD6I to a substrate. The optimal size of Nt.BspD6I’s binding site in DNA as determined in this work should be taken into account in methods of detection of nucleic acid sequences and/or even various base modifications by means of NEs.

Nucleic Acid Sequence Detection by a Multi-Technique Approach

The development of highly sensitive, specific, cheap and rapid laboratory diagnostics is an increasingly important challenge. Diagnosis of virus or various cancer diseases can be achieved through nucleic acid sequence detection, able to recognize specific short DNA or RNA sequences. DNA-based biosensors are excellent candidates, thanks also to their reusable property. A careful multi-technique characterization of DNA self-assembled monolayers (SAMs) represents a crucial step in order to optimize a sturdy sequence detection scheme based on helix-helix hybridization. In our work, we exploit molecular self-assembly on gold of single 22/28-bases DNA strands complementary to specific disease-related RNA or DNA sequences. The characterization is based on microscopic (Atomic Force Microscopy, AFM), spectroscopic (Spectroscopic Ellipsometry, SE, and X-rays Photoelectron Spectroscopy, XPS) and mass-sensitive (Quartz-Crystal Microbalance with Dissipation, QCM-D) methods, in order to investigate structural, optical and packing properties of DNA films. By AFM nanolithography, regularly defined micro-areas were depleted from molecules under a high tip load: the depth of the shaved area provides an accurate estimate of the film thickness. Specific sequence recognition has therefore been detected by monitoring changes in the film thickness upon hybridization. Through SE experiments it has been possible to monitor the optical thickness (influenced by both thickness and refractive index) and the fingerprints of the 260 nm DNA molecular absorption, allowing to evaluate the hybridization state of a DNA monolayer through a non-destructive and rapid method. Finally, XPS measurements provided useful information about surface coverage, allowing to distinguish between single strand and double strand DNA monolayers, while by QCM-D we could follow the dynamic process and, from the changes in mass/area ratio, evaluate the molecular density.

Biologically modified microelectrode sensors provide enhanced sensitivity for detection of nucleic acid sequences from Mycobacterium tuberculosis

This paper describes improved sensitivity when using biosensors based on microfabricated microelectrodes to detect DNA, with the goal of progressing towards a low cost and mass manufacturable assay for antibiotic resistance in tuberculosis (TB). The microelectrodes gave an improvement in sensitivity compared to polycrystalline macroelectrodes. In addition, experimental parameters such as redox mediator concentration and experimental technique were investigated and optimised. It was found that lower concentrations of redox mediator gave higher signal changes when measuring hybridisation events and, at these lower concentrations, square wave voltammetry was more sensitive and consistent than differential pulse voltammetry. Together, this paper presents a quantifiable comparison of macroelectrode and microelectrode DNA biosensors. The final assay demonstrates enhanced sensitivity through reduction of sensor size, reduction of redox mediator concentration and judicious choice of detection technique, therefore maintaining manufacturability for incorporation into point of care tests and lab-on-a-chip devices.

Simultaneous Nucleic Acids Detection and Elimination of Carryover Contamination With Nanoparticles-Based Biosensor- and Antarctic Thermal Sensitive Uracil-DNA-Glycosylase-Supplemented Polymerase Spiral Reaction.

The current report devised a novel isothermal diagnostic assay, termed as nanoparticle-based biosensor (NB)- and antarctic thermal sensitive uracil-DNA-glycosylase (ATSU)-supplemented polymerase spiral reaction (PSR; NB-ATSU-PSR). The technique merges enzymatic digestion of carryover contaminants and isothermal nucleic acid amplification technique (PSR) for simultaneous detection of nucleic acid sequences and elimination of carryover contamination. In particular, nucleic acid amplification and elimination of carryover contamination are conducted in a single pot and, thus, the use of a closed-tube reaction can remove undesired results due to carryover contamination. For demonstration purpose, Klebsiella pneumoniae is employed as the model to demonstrate the usability of NB-ATSU-PSR assay. The assay's sensitivity, specificity, and practical feasibility were successfully evaluated using the pure cultures and sputum samples. The amplification products were detectable from as little as 100 fg of genomic DNAs and from ~550 colony-forming unit (CFU) in 1 ml of spiked sputum samples. All K. pneumoniae strains examined were positive for NB-ATSU-PSR detection, and all non-K. pneumoniae strains tested were negative for the NB-ATSU-PSR technique. The whole process, including rapid template preparation (20 min), PSR amplification (60 min), ATSU treatment (5 min), and result reporting (within 2 min), can be finished within 90 min. As a proof-of-concept methodology, NB-ATSU-PSR technique can be reconfigured to detect various target nucleic acid sequences by redesigning the PSR primer set.

Colorimetric detection of nucleic acid sequences in plant pathogens based on CRISPR/Cas9 triggered signal amplification.

A colorimetric method is presented for the detection of specific nucleotide sequences in plant pathogens. It is based on the use of CRISPR/Cas9-triggered isothermal amplification and gold nanoparticles (AuNPs) as optical probes. The target DNA was recognized and broken up by a given Cas9/sgRNA complex. After isothermal amplification, the product was hybridized with oligonucleotide-functionalized AuNPs. This resulted in the aggregation of AuNPs and a color change from wine red to purple. The visual detection limit is 2 pM of DNA, while a linear relationship exists between the ratio of absorbance at 650 and 525nm and the DNA concentration in the range from 0.2 pM to 20nM. In contrast to the previous CRISPR-based amplification platforms, the method has significantly higher specificity with the single-base mismatch and can be visually read out. It was successfully applied to identify the Phytophthora infestans genomic DNA. Graphical abstract Schematic presentation of a colorimetric method for detection of Phytophthora infestans genomic DNA based on CRISPR/Cas9-triggered isothermal amplification. The Cas9 endonuclease cleaves DNA at the design site and the color changes from red to purple with increasing target DNA concentration.

Rift Valley Fever Viral RNA Detection by In Situ Hybridization in Formalin-Fixed, Paraffin-Embedded Tissues.

Sporadic outbreaks of Rift Valley fever virus (RVFV), a zoonotic, mosquito-borne Phlebovirus, cause abortion storms and death in sheep and cattle resulting in catastrophic economic impacts in endemic regions of Africa. More recently, with changes in competent vector distribution, growing international trade, and its potential use for bioterrorism, RVFV has become a transboundary animal disease of significant concern. New and sensitive techniques that determine RVFV presence, while lessening the potential for environmental contamination and human risk, through the use of inactivated, noninfectious samples such as formalin-fixed, paraffin-embedded (FFPE) tissues are needed. FFPE tissue in situ hybridization (ISH) enables the detection of nucleic acid sequences within the visual context of cellular and tissue morphology. Here, we present a chromogenic pan-RVFV ISH assay based on RNAscope® technology, which is able to detect multiple RVFV strains in FFPE tissues, enabling visual correlation of RVFV RNA presence with histopathologic lesions.

Generation and Single-Molecule Characterization of a Sequence-Selective Covalent Cross-Link Mediated by Mechlorethamine at a C–C Mismatch in Duplex DNA for Discrimination of a Disease-Relevant Single Nucleotide Polymorphism

Many strategies for the detection of nucleic acid sequence rely upon Watson-Crick hybridization of a probe strand to the target strand, but the reversible nature of nucleic acid hybridization presents an inherent challenge: short probes that provide high target specificity have relatively low target affinity resulting in signal losses. Sequence-specific covalent cross-linking reactions have the potential to provide both selective target capture and durable signal. We explore a novel approach involving sequence-specific covalent cross-linking of a probe to target DNA combined with single-molecule nanopore detection of the cross-linked DNA. Here, we exploited the selective reaction of mechlorethamine at a C-C mismatch for covalent capture of a target DNA sequence corresponding to a cancer-driving mutation at position 1799 of the human BRAF kinase gene. We then demonstrated that the α-hemolysin protein nanopore can be employed for the unambiguous, single-molecule detection of the cross-linked probe-target complex. Cross-linked DNA generates an unmistakable deep and persistent current block (≥5 s) that is easily distinguished from the microsecond and millisecond blocks generated by translocation of single-stranded DNA and uncross-linked duplexes through the nanopore.

Antarctic thermolabile uracil-DNA-glycosylase-supplemented multiple cross displacement amplification using a label-based nanoparticle lateral flow biosensor for the simultaneous detection of nucleic acid sequences and elimination of carryover contamination

Here, we report a novel and universal methodology, termed “Antarctic thermolabile uracil-DNA-glycosylase (AUDG)-supplemented nucleic acid amplification techniques (NAAs) using a labeled-based nanoparticle lateral flow biosensor (LFB)” (AUDG-NAAs-LFB), which merges enzymatic (AUDG) digestion of contaminant amplicons with different nucleic acid amplification techniques (NAAs), and uses a lateral flow biosensor (LFB) for the rapid and visual confirmation of the presence of a target nucleic acid sequence. AUDG-NNAs-LFB is a one-pot, closedvessel assay, that can effectively eliminate false-positive signals arising from either carryover contaminants or the interaction between labeled primers. A new LFB was devised for detecting three targets (two amplicons generated from amplification of target sequences, and a chromatography control), without the need for probe-hybridization or additional incubation steps. As a proof of concept, multiple cross displacement amplification (MCDA), which is a specific, sensitive, and rapid isothermal amplification method, was selected as the model amplification technique to demonstrate the feasibility of AUDG-NAAs-LFB. As a result, we demonstrate the applicability of the AUDG-MCDA-LFB method for simultaneously detecting high-risk human papillomaviruses genotypes 16 and 18, which are the most and second-most prevalent strains of the virus reported in women worldwide. We also confirm the principle behind the AUDG-MCDA-LFB assay and validate its sensitivity, reproducibility, and specificity using serial dilutions of the type-specific plasmids, as well as clinical samples. This proof-of-concept method (AUDG-MCDA-LFB) can be easily reconfigured to detect various nucleic acid sequences by redesigning the specific MCDA primers.

Multiplexed isothermal nucleic acid amplification

Multiplexed isothermal amplification and detection of nucleic acid sequences and biomarkers is of increasing importance in diverse areas including advanced diagnostics, food quality control and environmental monitoring. Whilst there are several very elegant isothermal amplification approaches, multiplexed amplification remains a challenge, requiring careful experimental design and optimisation, from judicious primer design in order to avoid the formation of primer dimers and non-specific amplification, applied temperature as well as the ratio and concentration of primers. In this review, we describe the various approaches that have been reported to date for multiplexed isothermal amplification, for both “one-pot” multiplexing as well as parallelised multiplexing using loop-mediated isothermal amplification, strand-displacement amplification, helicase-dependent amplification, rolling circle amplification, nucleic acid sequence-based amplification, with a particular focus on recombinase polymerase amplification.