DNA amplification is required for most molecular diagnostic applications, but conventional polymerase chain reaction (PCR) has disadvantages for field testing. Isothermal amplification techniques are being developed to respond to this problem. One of them is the recombinase polymerase amplification (RPA) that operates at isothermal conditions without sacrificing specificity and sensitivity in easy-to-use formats. In this work, RPA was used for the optical detection of solid-phase amplification of the potential biowarfare agent Yersinia pestis. Thiolated forward primers were immobilized on the surface of maleimide-activated microtitre plates for the quantitative detection of synthetic and genomic DNA, with elongation occurring only in the presence of the specific template DNA and solution phase reverse primers. Quantitative detection was achieved via the use of biotinylated reverse primers and post-amplification addition of streptavidin-HRP conjugate. The overall time of amplification and detection was less than 1 h at a constant temperature of 37 °C. Single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) sequences were detected, achieving detection limits of 4.04*10(-13) and 3.14*10(-16) M, respectively. The system demonstrated high specificity with negligible responses to non-specific targets.
Development of a fully automated electrochemiluminescence (ECL) DNA assay for multiplex detection of six biowarfare agents is described. Aminated-DNA capture probes were covalently immobilised on activated-carbon electrodes and subsequently hybridised to target strands. Detection was achieved via a sandwich-type assay after Ru(bpy)3(2+)-labelled reporter probes were hybridised to the formed probe-target complexes. The assay was performed in an automated microsystem in a custom designed ECL detection box with integrated fluidics, electronics,and movable photomultiplier detector. The obtained limits of detection were 0.6-1.2 nmol L(-1) for six targets ranging from 50 to 122 base pairs in size, with linear range 1-15 nmol L(-1). Non-specific adsorption and cross-reactivity were very low. Detection of six targets on a single chip was achieved with subnanomolar detection limits.
In November 2012, a group of 7 persons who participated in a hare hunt in North Rhine-Westphalia, Germany, acquired tularemia. Two F. tularensis subsp. holarctica isolates were cultivated from human and hare biopsy material. Both isolates belonged to the FTN002–00 genetic subclade (derived for single nucleotide polymorphisms B.10 and B.18), thus indicating likely hare-to-human transmission.
An electrochemiluminescence (ECL) immunosensor for the rapid detection of the Francisella tularensis pathogen using whole antibodies or antibody fragments as capture biomolecule is described. A sandwich immunoassay was used with either lipopolysaccharide (LPS) or the whole inactivated bacterial cell (LVS) as a target, while Ru(bpy)3 (2+)-encapsulated silicate nanoparticles were linked to the secondary antibody and used as ECL labels. The assay was performed in a fluidic chip housed in a custom-built black box incorporating electronics, optics and fluidics. The obtained limit of detection for LPS was 0.4 ng/mL, while for the LVS it was 70 and 45 bacteria/mL when the capturing molecule was the whole antibody and the antibody F(ab) fragment, respectively.
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