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PubMed2025

Using CRISPR for viral nucleic acid detection.

Pathogenic microorganisms, such as viruses, have threatened human health and will continue to contribute to future epidemics and pandemics, highlighting the importance of developing effective diagnostics. To contain viral outbreaks within populations, fast and early diagnosis of infected individuals is essential. Although current standard methods are highly sensitive and specific, like RT-qPCR, some can have slow turnaround times, which can hinder the prevention of viral transmission. The discovery of CRISPR-Cas systems in bacteria and archaea initially revolutionized the world of genome editing. Intriguingly, CRISPR-Cas enzymes also have the ability to detect nucleic acids with high sensitivity and specificity, which sparked the interest of researchers to also explore their potential in diagnosis of viral pathogens. In particular, the CRISPR-Cas13 system has been used as a tool for detecting viral nucleic acids. Cas13's capability to detect both target RNA and non-specific RNAs has led to the development of detection methods that leverage these characteristics through designing specific detection read-outs. Optimization of viral sample collection, amplification steps and the detection process within the Cas13 detection workflow has resulted in assays with high sensitivity, rapid turnaround times and the capacity for large-scale implementation. This review focuses on the significant innovations of various CRISPR-Cas13-based viral nucleic acid detection methods, comparing their strengths and weaknesses while highlighting Cas13's great potential as a tool for viral diagnostics.

CRISPR-Cas Systems↗

PubMed2005

Multilabeled pyrene-functionalized 2'-amino-LNA probes for nucleic acid detection in homogeneous fluorescence assays.

Homogeneous fluorescence assays for detection of nucleic acids are widely used in biological sciences. Typically, probes such as molecular beacons that rely on distance-dependent fluorescence quenching are used for such assays. Less attention has been devoted to tethering a single kind of fluorophores to oligonucleotides and exploiting hybridization-induced modulation of fluorescence intensity for nucleic acid detection. Herein, thermal denaturation experiments and fluorescence properties of oligodeoxyribonucleotides containing one or more 2'-N-(pyren-1-yl)carbonyl-2'-amino-LNA monomer(s) X are described. These pyrene-functionalized 2'-amino-LNAs display large increases in thermal stability against DNA/RNA complements with excellent Watson-Crick mismatch discrimination. Upon duplex formation of appropriately designed 2'-N-(pyren-1-yl)carbonyl-2'-amino-LNA probes and complementary DNA/RNA, intensive fluorescence emission with quantum yields between 0.28 and 0.99 are observed. Quantum yields of such magnitudes are unprecedented among pyrene-labeled oligonucleotides. Molecular modeling studies suggest that the dioxabicyclo[2.2.1]heptane skeleton and amide linkage of monomer X fix the orientation of the pyrene moiety in the minor groove of a nucleic acid duplex. Interactions between pyrene and nucleobases, which typically lead to quenching of fluorescence, are thereby reduced. Duplexes between multiple modified probes and DNA/RNA complements exhibit additive increases in fluorescence intensity, while the fluorescence of single stranded probes becomes increasingly quenched. Up to 69-fold increase in fluorescence intensity (measured at lambda(em) = 383 nm) is observed upon hybridization to DNA/RNA. The emission from duplexes of multiple modified probes and DNA/RNA at concentrations down to less than 500 nM can easily be seen by the naked eye using standard illumination intensities.

DNA↗

PubMed1999

Nucleic acid detection technologies -- labels, strategies, and formats.

Currently, no consensus exists on assay formats, labels, or detection reactions for nucleic acid assays. New labels continue to be developed and tested, and recent candidates include acetate kinase, firefly luciferase, and genes for enzymes. An additional trend is toward nonamplification strategies (e.g., branched chain and dendrimer type assays) as alternatives to the popular PCR and related amplification strategies. The new wave of microanalytical devices (microchips, with nanoliter to microliter internal volumes), massively parallel simultaneous test arrays, and the desire to produce hand-held sensors present new challenges and requirements for nucleic acid detection methods (e.g., analysis of large arrays of micrometer-sized spots of nucleic acid with high resolution). Here I review selected developments and new directions in nucleic acid assays.

DNA Probes↗

PubMed2005

Applications of Luminex xMAP technology for rapid, high-throughput multiplexed nucleic acid detection.

BACKGROUND: As we enter the post-genome sequencing era and begin to sift through the enormous amount of genetic information now available, the need for technologies that allow rapid, cost-effective, high-throughput detection of specific nucleic acid sequences becomes apparent. Multiplexing technologies, which allow for simultaneous detection of multiple nucleic acid sequences in a single reaction, can greatly reduce the time, cost and labor associated with single reaction detection technologies. METHODS: The Luminex xMAP system is a multiplexed microsphere-based suspension array platform capable of analyzing and reporting up to 100 different reactions in a single reaction vessel. This technology provides a new platform for high-throughput nucleic acid detection and is being utilized with increasing frequency. Here we review specific applications of xMAP technology for nucleic acid detection in the areas of single nucleotide polymorphism (SNP) genotyping, genetic disease screening, gene expression profiling, HLA DNA typing and microbial detection. CONCLUSIONS: These studies demonstrate the speed, efficiency and utility of xMAP technology for simultaneous, rapid, sensitive and specific nucleic acid detection, and its capability to meet the current and future requirements of the molecular laboratory for high-throughput nucleic acid detection.

Animals↗

PubMed2004

Labelfree fully electronic nucleic acid detection system based on a field-effect transistor device.

The labelfree detection of nucleic acid sequences is one of the modern attempts to develop quick, cheap and miniaturised hand-held devices for the future genetic testing in biotechnology and medical diagnostics. We present an approach to detect the hybridisation of DNA sequences using electrolyte-oxide-semiconductor field-effect transistors (EOSFETs) with micrometer dimensions. These semiconductor devices are sensitive to electrical charge variations that occur at the surface/electrolyte interface, i.e. upon hybridisation of oligonucleotides with complementary single-stranded (ss) oligonucleotides, which are immobilised on the oxide surface of the transistor gate. This method allows direct, time-resolved and in situ detection of specific nucleic acid binding events without any labelling. We focus on the detection mechanism of our sensors by using oppositely charged polyelectrolytes (PAH and PSS) subsequently attached to the transistor structures. Our results indicate that the sensor output is charge sensitive and distance dependent from the gate surface, which pinpoints the need for very defined surface chemistry at the device surface. The hybridisation of natural 19 base-pair sequences has been successfully detected with the sensors. In combination with nano-transistors a PCR free detection system might be feasible in future.

Biosensing Techniques↗

PubMed2001

Strategies for signal amplification in nucleic acid detection.

Many aspects of molecular genetics necessitate the detection of nucleic acid sequences. Current approaches involving target amplification (in situ PCR, Primed in situ Labeling, Self-Sustained Sequence Replication, Strand Displacement Amplification), probe amplification (Ligase Chain Reaction, Padlock Probes, Rolling Circle Amplification) and signal amplification (Tyramide Signal Amplification, Branched DNA Amplification) are summarized in the present review, together with their advantages and limitations.

DNA Probes↗

PubMed2003

Fluorescence energy transfer between fluorescein label and DNA intercalators to detect nucleic acids hybridization in homogeneous media.

A general approach to detecting nucleic acid sequences in homogeneous media by means of steady-state fluorescence measurements is proposed. The methodology combines the use of a fluorescence-labeled single-strand DNA model probe, the complementary single-strand DNA target, and a DNA intercalator. The probe was fluorescein labeled to a spacer arm at the N4 position of the cytosine amino groups in polyribocytidylic acid (5'), poly(C), which acts as a model DNA probe. The complementary strand was polyriboinosinic acid (5'), poly(I), as a model of the target, and the energy transfer acceptor was an intercalator, either ethidium bromide or ethidium homodimer. In previous papers we have shown that the fluorescence intensity of the fluorescein label decreases when labeled poly(C) hybridizes with poly(I), and this fluorescence quenching can be used to detect DNA hybridization or renaturation in homogeneous media. In this paper we demonstrate that fluorescence resonance energy transfer (FRET) between fluorescein labeled to poly(C) and an intercalator agent takes place when single-stranded poly(C) hybridizes with poly(I), and we show how the fluorescence energy transfer further decreases the steady-state fluorescence intensity of the label, thus increasing the detection limit of the method. The main aim of this work was to develop a truly homogeneous detection system for specific nucleic acid hybridization in solution using steady-state fluorescence and FRET, but with the advantage of only having to label the probe with the energy donor since the energy acceptor is intercalated spontaneously. Moreover, the site label is not critical and can be labeled randomly in the DNA strand. Thus, the method is simpler than those published previously based on FRET. The experiments were carried out in both direct and competitive formats.

Base Sequence↗

PubMed1990

The application of enhanced chemiluminescence to membrane-based nucleic acid detection.

The ECL gene detection system is a novel, sensitive, non-radioactive system for the detection of nucleic acid hybridized on both nylon and nitrocellulose membranes. It is characterized by direct labeling of probe sequences with horseradish peroxidase combined with an enhanced chemiluminescent (ECL) detection reaction; the light output is captured on blue-light sensitive film. The application of the system to a range of standard molecular biology hybridization techniques is described.

Blotting, Northern↗

PubMed1999

Ultrastructural methods for nucleic acid detection by immunocytology.

In the present review are summarized recent developments in immunocytochemical detection of nucleic acids in biological materials at the ultrastructural level. Not only the approaches using antibodies to natural nucleic acids are described but also the techniques involving the use of antibodies raised against various nucleotide analogs incorporated beforehand into nucleic acids. Special emphasis is placed on each method's potential and limitations. These methods, combined or not with molecular biotechnology, are powerful tools for studying the structure and function of nucleic acids. They can be used to investigate the distribution and topological organization of DNA and RNA molecules or of specialized within these molecules in the cells.

Animals↗

PubMed1994

Monoclonal antibodies targeted to alpha-oligonucleotides. Characterisation and application in nucleic acid detection.

The aim of the present study was to test the antigenicity of alpha-deoxyribonucleotides in order to develop a new tool for the detection of nucleic acid sequences for use in diagnostic applications. We describe four monoclonal antibodies (Mabs) which recognize alpha-deoxyribonucleotides. Two were raised against a poly(alpha-dT) sequence and specifically recognized the alpha-dT nucleotide. Two were raised against a sequence containing all four common nucleotides as alpha-nucleotides and, surprisingly, only recognized the alpha-dG nucleotide. For all four Mabs, no cross reactivity was observed with beta-oligonucleotides. These Mabs were reactive with alpha-oligonucleotide sequences whether these sequences were single-stranded or hybridized to DNA or RNA. The four Mabs were tested in a sandwich hybridization assay that consisted of an alpha-oligonucleotide (for target sequence recognition), one of the four Mabs (for recognition of the hybridized alpha-oligonucleotide), and goat anti-mouse antibody conjugated to horse radish peroxidase (HRP) (for detection). One of the monoclonal antibodies, Mab 2E11D7, was directly conjugated to HRP and used in sandwich hybridization to detect PCR fragments of HPV 18 DNA. The sensitivity of this reaction was 1 pg of plasmid DNA containing the HPV 18 fragment. The specificity of the detection was demonstrated using HPV 6/11 and 16 DNA sequences.

Animals↗

PubMed2000

Light-up probes: thiazole orange-conjugated peptide nucleic acid for detection of target nucleic acid in homogeneous solution.

We have constructed light-up probes for nucleic acid detection. The light-up probe is a peptide nucleic acid (PNA) oligonucleotide to which the asymmetric cyanine dye thiazole orange (TO) is tethered. It combines the excellent hybridization properties of PNA and the large fluorescence enhancement of TO upon binding to DNA. When the PNA hybridizes to target DNA, the dye binds and becomes fluorescent. Free probes have low fluorescence, which may increase almost 50-fold upon hybridization to complementary nucleic acid. This makes the light-up probes particularly suitable for homogeneous hybridization assays, where separation of the bound and free probe is not necessary. We find that the fluorescence enhancement upon hybridization varies among different probes, which is mainly due to variations in free probe fluorescence. For eight probes studied the fluorescence quantum yield at 25 degrees C in the unbound state ranged from 0.0015 to 0.08 and seemed to depend mainly on the PNA sequence. The binding of the light-up probes to target DNA is highly sequence specific and a single mismatch in a 10-mer target sequence was readily identified.

Base Pair Mismatch↗

PubMed1999

Interaction of cyanine dyes with nucleic acids. XII.beta-substituted carbocyanines as possible fluorescent probes for nucleic acids detection.

Results of investigations of fluorescent properties of a beta-substituted carbocyanine and its complexes with nucleic acids in comparison with those for the unsubstituted dye are presented. Carbocyanine substituted in polymethine chain has shown promising properties for use as a fluorescent probe in homogeneous systems of nucleic acids detection.

Carbocyanines↗

PubMed1997

Evaluation of commercially available tests for Chlamydia nucleic acid detection in synovial fluid of patients.

Since the presence of Chlamydia nucleic acids has been shown in synovial fluid (SF) from some patients with Chlamydia reactive arthritis, we investigated whether commercially available tests, developed to detect Chlamydia nucleic acids in urogenital samples, could also be used for their detection in SF samples. We therefore tested SF samples, found positive with at least two different systems of DNA amplification in a previous study, with three commercially available kits. No positive results were obtained. It is concluded that the commercially available tests Gen-Probe PACE 2, Amplicor (developed by Roche Molecular Systems) and LCx (developed by Abbott Laboratories) do not have sufficient sensitivity to detect reliably Chlamydia RNA or DNA in SF.

Antibodies, Bacterial↗

PubMed1995

Nucleic acid detection using non-radioactive labelling methods.

Nucleic acid probe-based assays are now widely used in genetic research, human identification, forensics and in a broad spectrum of clinical assays in the fields of microbiology, haematology/oncology and virology. Labelled probes are used in a variety of assay formats including dot-blots, Southern blots (DNA target), Northern blots (RNA target), Western blots (protein target), in situ hybridization, plaque or colony screening and immobilized arrays on silicon or glass surfaces. Traditionally, the probes used in these assays have a radioactive 32phosphorous label that has a short shelf-life, is dangerous, has high disposal costs and, when labelled to high specific-activity, may be unstable. Extensive efforts to develop alternative labelling techniques have resulted in colorimetric, chemiluminescent and fluorescent assay formats. This review summarizes the properties desired in a probe, describes the advantages and disadvantages of the different non-radioactive labelling strategies, and illustrates examples of probe-based assays in which detection is facilitated by imaging samples using a general purpose fluorescence scanner.

Colorimetry↗

PubMed2005

Universal labeling chemistry for nucleic acid detection on DNA chips.

An efficient strategy for nucleic acid labeling and analysis on deoxyribonucleic acid (DNA) chips has been developed. This approach, which combines the fragmentation and the labeling steps, is based on the reactivity of the phosphates of DNA and ribonucleic acid (RNA) fragments and is using reporter molecules bearing a bromomethyl- or aryldiazomethane-reactive group. In this chapter, we describe the preparation of the reactive label and protocols for efficient labeling of any nucleic acid sequence, DNA or RNA, prior to their hybridization, detection, and analysis on DNA chips.

Nucleic Acid Hybridization↗

PubMed1999

PNA biosensors for nucleic acid detection.

Biosensor devices, based on the conversion of nucleic acid recognition reactions into useful electrical signals, offer considerable promise for DNA diagnostics. The unique hybridization properties of solution-phase PNA can be extrapolated onto transducer surfaces in connection with the design of remarkably specific DNA biosensors. This article reviews the development of PNA biosensors, and discusses common PNA-biosensing protocols along with their prospects in DNA biosensor technology.

Biosensing Techniques↗

PubMed2005

Guidelines for the use and interpretation of nucleic acid detection tests for Neisseria gonorrhoeae in Australia: a position paper on behalf of the Public Health Laboratory Network.

The Public Health Laboratory Network (PHLN) convened a workshop of Australian experts in Melbourne on 23 March 2005 to identify laboratory issues of relevance and suggest guidelines for use of nucleic acid detection tests (NADT) for diagnosis of gonorrhoea in Australia. The proceedings of that meeting were endorsed by the members of the PHLN and the Communicable Diseases Network of Australia. Given the present state of knowledge and experience of conditions currently existing in Australia, the following recommendations were made: Recommendation 1: Assays using detection of the cppB gene should not be used for either screening or supplemental assays. Recommendation 2: All in-house screening assays that are positive should also be positive on a reliable supplemental assay before a positive result is reported. Recommendation 3: All commercial screening assays that are positive should also be positive on a reliable supplemental assay before a positive result is reported. Recommendation 4: If a sample is positive in a screening assay but a suitable supplemental assay is negative, then the result should be reported as negative. Recommendation 5: Laboratories should ensure that the test combination they use would yield a positive predictive value of at least 90 per cent in a population with a prevalence of 1 per cent. Recommendation 6: For the purposes of test evaluation, as distinct from diagnostic testing, true positives be defined by meeting one or more of the following criteria: 1) culture positive using contemporary isolation and identification techniques; 2) positive result on NADTs directed to targets on three separate genes that are known to have discriminatory capacity; 3) sequencing of a gene known to separate gonococcal from non-gonococcal species. Recommendation 7: Inhibitor controls should be routinely included in all NADT. Recommendation 8: Cultures are the preferred test for samples from non-genital sites. If however it is necessary to perform a NADT, then more stringent criteria should be applied, and positive samples should meet the 'test evaluation' criteria for a 'true positive'. Recommendation 9: In order to properly assess the routine diagnostic system in Australia, the following quality assurance samples should be distributed in addition to the routine samples currently used: 1. cppB negative N. gonorrhoeae; 2. Non-gonococcal species known to cause false positive reactions: these should be dispatched both as a single species, as well as mixture with N. gonorrhoeae. In the latter circumstance, the non-gonococcal species should be present in 10-fold excess; 3. Urine samples: preferably a single patient sample, otherwise a spiked sample. 4. Validation panels should be made that include samples that are culture positive but PCR negative. True positive samples should also be made available. In addition, a process should be established for full phenotypic and genotypic characterisation of unidentified species that yield false positive results in NADT for gonococci. Recommendation 10: Strategies should be put in place to ensure that sufficient numbers of gonococcal isolates are obtained to allow reliable monitoring of antimicrobial resistance. Recommendation 11: Public health practitioners need to define the relevant populations that need to be targeted and identify any that require enhanced surveillance.

Anti-Bacterial Agents↗

PubMed1990

Nonradioactive nucleic acid detection by enhanced chemiluminescence using probes directly labeled with horseradish peroxidase.

The use of nucleic acid probes directly labeled with horseradish peroxidase for detection of single copy sequences on Southern blots of human genomic DNA by enhanced chemiluminescence is described. Of the target sequences, 6 x 10(5) molecules (1 amol) have been detected on blue sensitive film using exposures of up to 60 min and probes of 0.3-5.1 kb. The chemiluminescent signal quantified using a cooled charge coupled device (CCD) camera is proportional to probe length for DNA probes in the range 50-3571 bases. The enzyme has no significant effect on the stability of a DNA/DNA hybrid formed with a 3571-base probe and target as determined by increasing the stringency of posthybridization washes by decreasing the concentration of a monovalent cation (NaCl) and by a Tm analysis. The kinetics of DNA hybridization have been analyzed by a cooled CCD camera to provide quantitative data. Ten nanograms per milliliter of probe may be used for an overnight hybridization. Southern blots can be reprobed using a DNA probe for the same or a different sequence without the necessity of stripping off the previously bound probe.

Blotting, Southern↗