Nucleic acids detection assays

Nucleic acid (deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)) probes utilize labeled, ie, radioactive, enzymatic, or fluorescent, fragments of DNA or RNA (the probe) to detect complimentary DNA or RNA sequences in a sample. Because the probe is tailored for one specific nucleic acid, these assays are highly specific and very sensitive (45). 

Nucleic acid-based detection may be more specific and sensitive than immunological-based detection. Furthermore, the polymerase chain reaction (PCR) can be easily coupled to enhance the sensitivity of nucleic acid-based assays. Target nucleic segments of defined length... 

However, the nucleic acid-based assays for the detection of food pathogens show problems regarding the sensitivity of the polymerase enzyme to environmental contaminants, difficulties in quantification, the generation of false-positives through the detection of naked nucleic acids, non-viable microorganisms or contamination of samples in the laboratory, and may limit the use of PCR for the direct detection of microbial contamination. 

Solution-based nucleic acid hybridization assays represent a class of analytical methodologies that provide for detection of target-probe hybridization events. Solution-based assays offer several advantages in comparison to configurations that use surface-bound probes, primarily from the standpoint of negating the requirement for immobilization of a probe sequence to a solid surface. Therefore, thermodynamic, kinetic and adsorptive effects that are relevant in terms of consideration of hybridization at interfaces are not encountered. Solution-based hybridization assays provide a simple way of detecting hybridization events in real-time. 

The use of ECL processes for the detection of biological compounds is a rapidly growing area of interest, both for quantitation of analytes and to measure biomolecular interactions. By using ECL active chromophores as labels for biological compounds, a variety of applications is possible, including assays for enzymatic activity, binding assays, immunoassays, and nucleic acid probe assays. 

Enzyme DNA hybridization assays with electrochemical detection can offer enhanced sensitivity and reduced instrumentation costs in comparison with their optical counterparts. Efforts to prevent non-specific binding of the codissolved enzyme and to avoid fouling problems by selecting conditions suitable to amplify the electrode response have been reported by Heller and co-workers [107]. A disposable electrochemical sensor based on an ion-exchange film-coated screen-printed electrode was described by Limoges and co-workers for an enzyme nucleic acid hybridization assay using alkaline phosphatase [108] or horseradish peroxidase [109]. In another methodology to improve sensitivity, a carbon paste electrode with an immobilized nucleotide on the electrode surface and methylene blue as hybridization indicator was coupled, by Mascini and co-workers [110], with PGR amplification of DNA extracted from human blood for the electrochemical detection of virus. 

Baker CA, Cartwright CP, Williams DN> Nelson SM, Peterson PK. Early detection of central nervous system tuberculosis with the gen-probe nucleic acid amplification assay utility in an inner city hospital. Clin Infect Dis 2002 35 339-42. 

Good assay reproducibility and recovery were observed for neat (undiluted) normal human serum and serum from rheumatoid arthritis patients for quantitation of a fully human anti-TNF-a monoclonal antibody [86]. It has also been reported that excess therapeutic antibodies present in serum were tolerated better in an assay for the detection of antitherapeutic antibodies based on the MSD ECL device [91]. Furthermore, the ECL procedure has been reported to be stable over a broad range of magnetic bead concentrations, probe concentrations, and hybridization conditions for a nucleic acid binding assay, making these assays more versatile and easier to transfer from one laboratory to another [88]. 

This article is designed to provide readers who are unfamiliar with chemiluminescence with a sufficient theoretical basis to understand the phenomenon and to extend that basis with a survey of its evolution in terms of chemistry, analytical applications, and recent innovations both in high sensitivity clinical assays and in nucleic acid detection methods. 

Immunoassay and nucleic acid probe assays Labels. recApoaequorin, firefly luciferase, marine bacterial luciferase, Vargula luciferase Detection reactions, alkaline phosphatase label (luciferin-O-phosphate/firefly luciferase), glucose 6-phosphate dehydrogenase label (marine bacterial luciferase/NADH FMN oxidoreductase reaction)... 

Laschi S, Miranda-Castro R, Gonz ez-Femandez E, Palchetti I, Reymond F, Rossier JS, Marrazza G (2010) A new gravity-driven microfluidic-based electrochemical assay coupled to magnetic beads for nucleic acid detection. Electrophoresis 31 1-10... 

Assays based on nucleic acid detection are now becoming commonplace in the diagnostic market scene. For example, DNA and RNA based diagnostic kits for detection of viruses and pathogenic organisms are commercially available from companies such as Gen-Probe [9], Chiron [10], and Digene [11]. Nucleic acids are... 

Yeh, H. Ho, Y. Wang, T. Quantum dot-mediated biosensing assays for specific nucleic acid detection. Nanomedicine 2005,1,115-121. 

As the result of high specificity and sensitivity, nucleic acid probes are in direct competition with immunoassay for the analytes of some types of clinical analytes, such as infectious disease testing. Assays are being developed, however, that combine both probe and immunoassay technology. In such hybrid probe—immunoassays, the immunoassay portion detects and amplifies the specific binding of the probe to a nucleic acid. Either the probe per se or probe labeled with a specific compound is detected by the antibody, which in turn is labeled with an enzyme or fluorophore that serves as the basis for detection. 

The emission yield from the horseradish peroxidase (HRP)-catalyzed luminol oxidations can be kicreased as much as a thousandfold upon addition of substituted phenols, eg, -iodophenol, -phenylphenol, or 6-hydroxybenzothiazole (119). Enhanced chemiluminescence, as this phenomenon is termed, has been the basis for several very sensitive immunometric assays that surpass the sensitivity of radioassay (120) techniques and has also been developed for detection of nucleic acid probes ia dot-slot. Southern, and Northern blot formats (121). 

Current analytical methods have difficulty detecting picogram levels of nucleic acids, particularly when high levels of other biopolymers (e.g., proteins) are present. The most widely used assay method employed by the pharmaceutical industry involves a nick translation DNA hybridization method (1). This assay offers high sensitivity and selectivity but has a number of drawbacks. 

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