Real-time PCR quantification of the AM-toxin gene and HPLC qualification of toxigenic metabolites from Alternaria species from apples.

Some Alternaria species are able to produce plant pathogenic as well as toxic metabolites. In both agriculture and the food industry it is important know if toxigenic Alternaria are present to rapidly employ the correct corrective actions. The purpose of this work was to establish a real-time PCR method, which can detect and quantify apple pathogenic and toxigenic Alternaria. An AM-toxin I primer set, which could recognize Alternaria DNA only, was designed by using primers complementary to the AM-toxin I gene. The method could detect small amounts of DNA (4 pg) and still obtain a large dynamic range (4 decades) without interference from apple material. Eight Alternaria isolates were analyzed for the presence of AM-toxin I gene and their production of secondary metabolites. Then analyses showed that all eight isolates contained the AM toxin gene and were able to produce the plant pathogenic tentoxin in addition to AM toxin I. The analyses also showed the production of tenuazonic acid, alternariols, Altenuene, altenusin and/or altertoxin I in pure culture. Analyses of inoculated apples showed that both the AM-toxin gene and alternariol monomethyl ether could be detected. Morphological analyses suggested that the eight Alternaria strains, though they all carried the AM toxin genes, probably belong to different but closely related un-described Alternaria taxa in the A. tenuissima species-group based on morphological and chemical differences.

Towards biochips using microstructured optical fiber sensors.

In this paper we present the first incorporation of a microstructured optical fiber (MOF) into biochip applications. A 16-mm-long piece of MOF is incorporated into an optic-fluidic coupler chip, which is fabricated in PMMA polymer using a CO(2) laser. The developed chip configuration allows the continuous control of liquid flow through the MOF and simultaneous optical characterization. While integrated in the chip, the MOF is functionalized towards the capture of a specific single-stranded DNA string by immobilizing a sensing layer on the microstructured internal surfaces of the fiber. The sensing layer contains the DNA string complementary to the target DNA sequence and thus operates through the highly selective DNA hybridization process. Optical detection of the captured DNA was carried out using the evanescent-wave-sensing principle. Owing to the small size of the chip, the presented technique allows for analysis of sample volumes down to 300 nL and the fabrication of miniaturized portable devices.

Photonic crystal fiber long-period gratings for biochemical sensing.

We present experimental results showing that long-period gratings in photonic crystal fibers can be used as sensitive biochemical sensors. A layer of biomolecules was immobilized on the sides of the holes of the photonic crystal fiber and by observing the shift in the resonant wavelength of a long-period grating it was possible to measure the thickness of the layer. The long-period gratings were inscribed in a large-mode area silica photonic crystal fiber with a CO2 laser. The thicknesses of a monolayer of poly-L-lysine and double-stranded DNA was measured using the device. We find that the grating has a sensitivity of approximately 1.4nm/1nm in terms of the shift in resonance wavelength in nm per nm thickness of biomolecule layer.