A real-time multiplex-nested PCR system for coprological diagnosis of Echinococcus multilocularis and host species

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.

Development of a Real-Time PCR for a Sensitive One-Step Coprodiagnosis Allowing both the Identification of Carnivore Feces and the Detection of Toxocara spp. and Echinococcus multilocularis

“…(21) and Echinococcus multilocularis (22,23) can be detected and quantified in stools. Real-time nested PCR using fluorescence resonance energy transfer (FRET) probes was designed to detect and discriminate between different host species by melting curve analysis for field studies focusing on Echinococcus multilocularis investigations and the carnivore hosts Vulpes vulpes (red fox), Vulpes ferrilata (Tibetan fox), and Canis lupus familiaris/Canis lupus (domestic dog/wolf) (22). However, in field studies dealing with helminth parasite detection and quantification, a larger panel of wild and domestic carnivores needs to be taken into account.…”

Molecular Diagnosis of Polycystic Echinococcosis Due to Echinococcus vogeli in a Paraguayan Immigrant in Argentina

“…This underlines the difficulty of DNA extraction from FFPE (degradation of DNA and low yield of extraction of long DNA fragments) and the need for shorter PCR targets for identification of infectious agents using PCR and sequencing in FFPE tissue (16)(17)(18)). An alternative quantitative PCR (qPCR) approach that targets short mitochondrial DNA fragments could also be of interest on FFPE DNA extract, either by combining species-specific targets or by defining genus-specific system, followed by melting curve analysis for species diagnosis (19,20). However, given the genetic variability of this parasite, the design of the specific primers and probes must be based on sequence data from a wide panel of E. vogeli samples (21).…”

Development of real-time PCR assay for simultaneous detection and genotyping of cystic echinococcosis in humans and livestock

“…In addition to the conventional PCR assays used for the detection of CE, most of the researchers have also utilized the nested amplification for the purpose of increasing the sensitivity and confirming the identity of the primary amplified PCR product [9,29,30]. Moreover, the amplified PCR requires digestion by restriction enzyme using PCR-RFLPs techniques for genotyping of EG strain, which is a rather expensive, laborious and time consuming technique [6,31].…”