Electrochemical Impedimetric Real-Time Polymerase Chain Reactions Using Anomalous Charge Transfer Enhancement.

We developed a new electrochemical impedimetric method for the real-time detection of polymerase chain reactions (PCR) based on our recent discovery that the DNA intercalator, [Ru(bpy)2DPPZ]2+, anomalously enhances charge transfer between redox mediators, K4[Fe(CN)6]/K3[Fe(CN)6], and a carbon electrode. Three mM [Fe(CN)6]3-/4- and 5 µM [Ru(bpy)2DPPZ]2+ were added to the PCR solution, and electrochemical impedance spectroscopy (EIS) measurements were performed at each elongation heat cycle. The charge transfer resistance (Rct) was initially low due to the presence of [Ru(bpy)2DPPZ]2+ in the solution. As PCR progressed, amplicon dsDNA was produced exponentially, and intercalated [Ru(bpy)2DPPZ]2+ ions, which could be detected as a steep Rct, increased at specific heat cycles depending on the amount of template DNA. The Rct increase per heat cycle, ΔRct, showed a peak at the same heat cycle as optical detection, proving that PCR can be accurately monitored in real time by impedance measurement. This simple method will enable a cost-effective and portable PCR device.

Identification of Staphylococcus spp. and detection of mecA by an oligonucleotide array.

Phenotypic identification of coagulase-negative staphylococci (CoNS) is difficult and many staphylococcal species carry mecA. This study developed an array that was able to detect mecA and identify 30 staphylococcal species by targeting the internal transcribed spacer regions. A total of 129 target reference strains (30 species) and 434 clinical isolates of staphylococci were analyzed. Gene sequencing of 16S rRNA, gap or tuf genes was the reference method for species identification. All reference strains (100%) were correctly identified, while the identification rates of clinical isolates of S. aureus and CoNS were 98.9% and 98%, respectively. The sensitivity and specificity for mecA detection were 99% and 100%, respectively, in S. aureus isolates, and both values were 100% in isolates of CoNS. The assay takes 6 h from a purified culture isolate, and so far it has not been performed directly on patient samples.

Optimization of the score cutoff value for routine identification of Staphylococcus species by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry.

Staphylococcus species are important pathogens. We evaluated 2 score cutoffs (2.0 and 1.7) and the replicate number (a single or a duplicate test) on the identification of staphylococci using the Bruker matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). A collection of 440 clinical isolates (11 species) and 144 reference strains (36 species) was evaluated. For clinical isolates using a cutoff of 2.0 and duplicate tests, the rates of species, genus, and unreliable identifications were 93.4%, 5.7%, and 0.9% respectively, while the respective values were 99.3%, 0.2%, and 0.5% when the cutoff was 1.7. The species identification rates were significantly higher (P<0.01) when a cutoff of 1.7 or a duplicate test was used. Similar results were obtained for reference strains. In conclusion, a cutoff of 1.7 and duplicate tests are recommended for identification of staphylococci using MALDI-TOF MS.

Rapid detection of dermatophytes and Candida albicans in onychomycosis specimens by an oligonucleotide array.

BACKGROUND: Onychomycosis is a fungal infection of nails, leading to the gradual destruction of the nail plate. Treatment of onychomycosis may need long-time oral antifungal therapy that can have potential side effects, thus accurate diagnosis of the disease before treatment is important. Culture for diagnosis of onychomycosis is time-consuming and has high false-negative rates. To expedite the diagnosis, an oligonucleotide array, based on hybridization between immobilized oligonucleotide probes and PCR products, for direct detection of dermatophytes and Candida albicans in clinical specimens was evaluated. METHODS: Species-specific oligonucleotide probes designed from the internal transcribed spacer (ITS) regions of the rRNA gene were immobilized on a nylon membrane. The assay procedures consisted of PCR amplification of the ITS using universal primers, followed by hybridization of the digoxigenin-labeled amplicons to probes on the array. Thirty two nail samples (29 patients) were analyzed by the array, and the results were compared with those obtained by culture. Array-positive but culture-negative samples were confirmed by cloning and re-sequencing of the amplified ITS and by reviewing patient's clinical data. The total recovery of culture and confirmed array-positive but culture-negative results was considered 100% and was used for performance evaluation of both methods. RESULTS: Concordant results were obtained in 21 samples (10 positives and 11 negatives) by both methods. Eleven samples were array-positive but culture-negative; among them, 9 samples were considered true positives after discrepant analysis. Comparing with culture, the array had significantly higher sensitivity [100% (95% CI 82.2% -100%) vs 52.6% (28.9% -75.5%), p <0.001] and negative predictive value [100% (71.3% -100%) vs 59.1% (36.4% -79.3%), p <0.05), while no significant differences were observed in specificity (84.6% vs 100%, p =0.48) and positive predictive value (90.5% vs 100%, p =1.0). The whole procedures of the array were about 24 h, whilst results from culture take 1 to 3 weeks. CONCLUSIONS: The array offers an accurate and rapid alternative to culture. Rapid diagnosis can expedite appropriate antifungal treatment of onychomycosis. However, the single site nature of this study conducted at a referral hospital invites caution.

Dielectrophoresis and shear-enhanced sensitivity and selectivity of DNA hybridization for the rapid discrimination of Candida species.

We present a dielectrophoresis (DEP)-based microfluidic chip that is capable of enhancing the sensitivity and selectivity of DNA hybridization using an AC electric field and hydrodynamic shear in a continuous through-flow. Molecular DEP was employed to rapidly trap ssDNA molecules in a flowing solution to a cusp-shaped nanocolloid assembly on a microfluidic chip with a locally amplified AC electric field gradient. The detection time can be accelerated to sub-minute periods, and the sensitivity can reach the pico-molar level due to the AC DEP-enhanced molecule concentration (at an optimal AC frequency of 900 kHz) in a small region (∼100 µm(2)) instead of the broad area used in a tank reactor (∼10(6) µm(2)). Continuous flow in a microchannel provides a constant and high shear rate that can shear off most non-specific target-probe binding to promote the discriminating selectivity. On-chip multi-target discrimination of Candida species can be achieved within a few minutes under optimal conditions.