ABSTRACT
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.
Subject(s)
Dog Diseases/diagnosis , Echinococcosis/veterinary , Echinococcus multilocularis/isolation & purification , Feces/parasitology , Parasitology/methods , Polymerase Chain Reaction/methods , Animals , China , Dog Diseases/parasitology , Dogs , Echinococcosis/diagnosis , Echinococcosis/parasitology , Sensitivity and SpecificityABSTRACT
The hybridisation characteristics of DNA targets to solid phase bound probes, e.g. in DNA microarrays, depend on the probe-target position and on target renaturation if a dsDNA target is used. We investigated a lambda exonuclease treatment of a PCR amplified dsDNA target to produce ssDNA with regard to probe-target position, treatment duration and inactivation time towards its impact on fluorescence or electrical signals on two DNA-chip formats. Surprisingly, the achieved amplification factors varied by three orders of magnitude, i.e. 2-1074 fold signal enhancement, depending on the relative probe-target position and readout scheme. The presented results can be used to design future studies involving lambda exonuclease preanalytic treatments.
