Simultaneous detection and differentiation of human rhino- and enteroviruses in clinical specimens by real-time PCR with locked nucleic Acid probes.

Human rhinoviruses (HRVs) and human enteroviruses (HEVs) are significant respiratory pathogens. While HRV infections are restricted to the respiratory tract, HEV infections may spread to secondary target organs. The method of choice for sensitive specific detection of these viruses is reverse transcription (RT)-PCR with primers targeting the conserved 5' noncoding region of the viral RNA. On the other hand, sequence similarities between HRVs and HEVs complicate their differential detection. In this study, we describe the use of locked nucleic acid (LNA) analogues in short double-dye probes which contained only two selectively HRV- or HEV-specific bases. The double-stranded DNA dye BOXTO (4-[6-(benzoxazole-2-yl-(3-methyl-)-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)]-1-methyl-quinolinium chloride) was used with the LNA probes in a tricolor real-time PCR assay to allow specific detection of HRVs (probes labeled with 6-carboxyfluorescein [FAM] [green]) and HEVs (Cy5 [red]) with additional melting curve analysis (BOXTO [yellow]). The functionality of the probes was validated in PCR and RT-PCR assays using plasmids containing viral cDNA, quantified viral RNA transcripts, cultivated rhino- and enterovirus prototypes, and clinical specimens. Of 100 HRV and 63 HEV prototypes, the probes correctly identified all HEVs except one that produced only a BOXTO signal. Among 118 clinical specimens with sequencing results, concordant results were obtained for 116 specimens. Two specimens were reactive with both probes, but sequencing yielded only a single virus. Real-time PCR with LNA probes allowed sensitive group-specific identification of HRVs and HEVs and would enable relative copy number determination. The assay is suitable for rapid and accurate differential detection of HRVs and HEVs in a diagnostic laboratory setting.

Time-resolved detection of hot electron-induced electrochemiluminescence of fluorescein in aqueous solution.

Strong electrogenerated chemiluminescence (ECL) of fluorescein is generated during cathodic pulse polarization of oxide-covered aluminum electrodes and the resulting decay of emission is so sluggish that time-resolved detection of fluorescein is feasible. The present ECL in aqueous solution is based on the tunnel emission of hot electrons into the aqueous electrolyte solution, which probably results in the generation of hydrated electrons and hydroxyl radicals acting as redox mediators. The successive one-electron redox steps with the primary radicals result in fluorescein in its lowest excited singlet state. The method allows the detection of fluorescein (or its derivatives containing usable linking groups to biomolecules) over several orders of magnitude of concentration with detection limits well below nanomolar concentration level. The detection limits can still be lowered, e.g., by addition of azide or bromide ions as coreactants. The results suggest that the derivatives of fluorescein, such as fluorescein isothiocyanate (FITC), can be detected by time-resolved measurements and thus be efficiently used as electrochemiluminescent labels in bioaffinity assays.

Rapid electrochemiluminoimmunoassay of human C-reactive protein at planar disposable oxide-coated silicon electrodes.

Electrochemiluminescence (ECL) of aromatic Tb(III) chelates at thin insulating film-coated electrodes provides a means for extremely sensitive detection of Tb(III) chelates and also of biologically interesting compounds if these chelates are used as labels in bioaffinity assays. The suitability of silicon electrodes coated with thermally grown silicon dioxide film as disposable working electrodes in sensitive time-resolved ECL measurements is demonstrated, and a rapid electrochemiluminoimmunoassay (ECLIA) of human C-reactive protein (hCRP) is described. Tb(III) chelate labels can be detected almost down to picomolar level, and the calibration curve of these labels covers more than 6 orders of magnitude of chelate concentration. The calibration curve of the present immunometric hCRP assay was found to be linear over a wide range, approximately 4 orders of magnitude of hCRP concentration, the detection limit of the protein being 0.3 ng mL(-1) (mean background + 2SD) on CV values of about 10-30%, depending on the immunoassay incubation time. In the ECLIA measurements, different incubation times were tested from 15 min (giving above-mentioned performance) to as short as only 2 min, which still gave successful results with approximately 20,000 times better detection limit levels than traditional commercial assay methods. During the ECLIA process, also the Si electrode surface morphology was also investigated by atomic force microscope monitoring.