Species detection within the Echinococcus granulosus sensu lato complex by novel probe-based Real-Time PCRs : [Preprint]
Background: Infections with eggs of Echinococcus granulosus sensu lato (s.l.) can cause cystic echinococcosis in intermediate host animals and humans. Upon ingestion of viable eggs, oncospheres hatch from the eggs and subsequently develop into fluid-filled larval cysts, most frequently in the liver or the lungs. The slowly growing cysts progressively interfere with organ function. The risk of infection is determined by the host range of the parasite, its pathogenicity and other epidemiologically relevant parameters, which differ significantly among the five species within the E. granulosus s.l. complex. It is therefore essential to diagnose the correct species within E. granulosus s.l. to help understand specific disease epidemiology and to facilitate effective implementation of control measures. For this purpose, simple, fast and cost-effective typing techniques are needed. Methods: Mitochondrial genome sequences from the respective members of E. granulosus s.l. complex were used to identify target regions for the detection and genotyping. The selected primer pairs were first tested in a SYBR-green assay. In the next step, primers discriminating between the genotypes (according to post-amplification melting curves) were tested together with the respective probes in a TaqMan® Real-Time Polymerase Chain Reactions (qPCRs). For the analysis of analytical sensitivity and specificity of the real-time PCRs (SYBR-green and TaqMan), a panel of reference DNAs from various Echinococcus spp. was used. Results: We developed quantitative qPCRs and corresponding sequence-specific hydrolysis DNA probes to target polymorphic regions in the mitochondrial genome of E. granulosus s.l.. In a single-step typing approach, we distinguished E. granulosus s.l. members in four epidemiologically relevant subgroups. These were E. granulosus sensu stricto (G1, G3), E. equinus (G4), E. ortleppi (G5) and the E. canadensis cluster (G6 to G8 and G10). The technique also allowed identification and differentiation of these species from other Echinococcus or Taenia taxa for samples isolated from cysts or faeces. Conclusions: Single-step genotyping techniques for the molecular diagnosis of Echinococcus spp. by qPCRs may not only improve diagnostic performance, but also our knowledge on the epidemiology of the parasites and help controlling the various agents of cystic echinococcosis.
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