Abstract / Summary
Echinococcus multilocularis is a zoonotic cestode capable of causing disease in humans and animals. Its eggs are released into the environment via the feces of definitive hosts and are the direct infectious source of human alveolar echinococcosis. Therefore, sensitive and species-specific monitoring of E. multilocularis egg contamination is crucial for effective disease control. We used our previously published single-tube nested minor groove binder (MGB) probe real-time PCR method, which includes specific probes for E. multilocularis and E. granulosus, to detect E. multilocularis eggs. The assay combines nested pre-amplification for analytical sensitivity with MGB probe detection for specificity in a closed-tube format, reducing contamination risk compared with conventional two-step nested PCR. Compared with coproscopy, it enables species-specific identification, and compared with conventional PCR, it provides detectable amplification at lower tested concentrations. The results showed no cross-reactivity with tested non-target canine parasites and exhibited intra-assay repeatability with coefficients of variation below 3%. The assay reagents remained stable under various storage conditions, including long-term storage at 4 °C and multiple freeze–thaw cycles, when tested with DNA extracted from eggs. In a preliminary field evaluation of 101 canine fecal samples, the assay and conventional PCR identified the same 12 positive samples, and all 12 were confirmed as E. multilocularis by sequencing. This assay provides a practical tool for further evaluation in epidemiological surveillance. However, its diagnostic sensitivity and specificity require further evaluation in larger field studies.