A hybridization probe-based real-time multiplex-nested PCR system was developed for the simultaneous detection of Echinococcus multilocularis and host species directly from faecal samples. Species identification was determined by melting curve analysis. Specificity was assessed by using DNA extracted from various cestodes (E. multilocularis, Echinococcus granulosus (G1), Echinococcus ortleppi, Echinococcus canadensis (G6, G7), Taenia crassiceps, Taenia hydatigena, Taenia mustelae, Taenia pisiformis, Taenia serialis, Taenia taeniaeformis, Mesocestoides leptothylacus), carnivores (Vulpes vulpes, Vulpes corsac, Vulpes ferrilata, Canis familiaris, Felis catus, Martes foina), Microtus arvalis and Arvicola terrestris. The analytical sensitivity was 10 fg, evaluated with serially diluted DNA of E. multilocularis to 10 μl total DNA solution from E. multilocularis-negative canid faeces. Based on a comparison of 47 dog samples from China, the proportion of the E. multilocularis-positive-tested samples by the real-time multiplex-nested PCR was moderately higher (38% vs. 30%) as when tested with a previously evaluated nested PCR with a sensitivity of 70-100%, depending on the number and gravidity status of worms present in the intestine (Dinkel et al., J Clin Microbiol 36:1871-1876, 1998). To assess the epidemiological applicability of this method, 227 canid faecal samples collected in the field were analysed. This newly developed real-time multiplex-nested PCR system is a specific, sensitive and reliable method for the detection of E. multilocularis and host species in faecal samples for epidemiological purposes.
Here we present a novel approach using surface-enhanced Raman scattering (SERS) spectroscopy for the sequence-specific detection of DNA utilizing magnetic nanoparticles (MNPs) for the enrichment of the target molecules. To achieve fast and efficient binding of longer DNA strands, e.g. PCR products, the hybridization procedure is performed in solution. To further purify and enrich the DNA strands of interest, MNPs are used for their separation. Following the binding of the target DNA, a dye-modified, short synthetic ssDNA is hybridized, which serves as label for the SERS detection. The SERS spectra are used to identify the bound molecules. The applicability of this approach was first tested with short synthetic oligonucleotides to evaluate its specificity. Afterward, the system was applied to detect PCR products amplified from DNA of specific agents of epizootic diseases. Sequences of the bacterium Mycoplasma mycoides subspecies mycoides small colony type (MmmSC), causing contagious bovine pleuropneumonia (CBPP) were used as PCR targets. To demonstrate the multiplexing capability of SERS, the simultaneous detection of three different PCR products labeled with three dyes was performed.
A portable and robust system which is suitable for the automated analysis of DNA or RNA of selected pathogens such as foot‐and‐mouth disease virus (FMDV) is developed. The system incorporates a stationary PCR chip and is coupled with a DNA chip and an electrical detection for the sequence‐specific identification of the PCR products. The PCR chip represents a miniaturized form of the classical thermocyclers and enables a fast and sensitive amplification as well as labeling of specific DNA sequences with minimal space and energy requirements. The detection and identification of the PCR products is performed on a DNA chip with an electrical detection scheme. The combination of the two technologies allows a very fast and highly specific sequence‐based detection and differentiation of pathogens. Further, it combines the accuracy of sequence analysis with the speed of chip technologies. For the total analysis including DNA amplification and DNA detection, less than 2 h are required.
Background: In gymnastics vaulting it is thought that gymnasts regulate their run-up on the basis of visually perceived environmental information, such as the position of the springboard, with the aim of an accurate foot placement on the springboard. The question, however, arises if these regulative processes found in gymnastics vaulting can be generalized to other tasks with similar demands but differing dynamics? Material/Methods: To answer this question, ten female gymnasts were asked to perform two target-directed gymnastics tasks that were similar in task demands but differed in task dy-namics. When performing the two tasks, the position of the springboard was manipulated without the gymnast’s awareness. Results: Results revealed that manipulating the position of the springboard had neither an effect on the distance of the hurdle, nor on the placement of the feet on the spring-board during the reactive leap. The two parameters, however, clearly differed between experimental tasks. Additionally, regulation during run-up occurred on average one step earlier when performing the tucked leap on the balance beam. Conclusions: It can be concluded from the results that gymnasts exhibit a different movement behavior when performing tasks with similar demands but different dynamics, thereby integrating environmental information in the regulation of the run-up and the reactive leap from trial to trial.
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