Echinococcus multilocularis (Leuckart, 1863)

Kjaer, Lene Jung, Jensen, Laura Mark, Chri, Marian, el, Ren, Bødker, e & Petersen, Heidi Huus, 2021, The raccoon dog (Nyctereutes procyonoides) as a reservoir of zoonotic diseases in Denmark, International Journal for Parasitology: Parasites and Wildlife 16, pp. 175-182 : 176-177

2.2.2. Echinococcus multilocularis

Genomic DNA was isolated from 0.25 g faeces using DNeasy PowerSoil Kit (Qiagen, Hilden, Germany) following the instructions of the manufacturer. Samples were analysed for the presence of E. multilocularis eggs using the E. multilocularis primer set EmSP1-A’/B ′

176

with forward primer 5 ′ -GTCATATTTGTTTAAGTATAAGTGG-3 ′ and reverse primer 5 ′ -CACTCTTATTTACACTAGAATTAAG-3’ ( Nonaka et al., 2009) targeting a fraction of the cytochrome c oxidase subunit I gene (cox1). For all reactions, mastermix were prepared containing all ingredients to ensure homogeneity between wells. All reactions in volumes of 22 μl, containing 0.7 μM of each primer, 5u/μl KAPA 2G robust DNA polymerase, 5.0 μl KAPA 2G buffer, 5.0 μl KAPA Enhancer (all KAPA 2G products were included in the KAPA 2G Robust PCR kit, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), 0.2 mM dNTP, 2.0 μl DNA and dH 2 O. The PCR was performed in a conventional PCR machine using the following conditions: 95 ◦ C for 15 min followed by 40 cycles of 95 ◦ C for 30 s, 50 ◦ C for 30 s, 72 ◦ C for 30 s and final extension for 10 min at 72 ◦ C. The protocol could consistently amplify DNA from two E. multilocularis eggs per 0.25 g faeces. A negative control without DNA was included in all tests.