Aelurostrongylus abstrusus (Railliet, 1898)

Lemming, Louise, Jørgensen, Ann Cholewa, Nielsen, Linette Buxbom, Nielsen, Stine Thorsø, Mejer, Helena, Chri, Mariann, el & Petersen, Heidi Huus, 2020, Cardiopulmonary nematodes of wild carnivores from Denmark: Do they serve as reservoir hosts for infections in domestic animals?, International Journal for Parasitology: Parasites and Wildlife 13, pp. 90-97 : 92

2.4.2. Molecular identification of Aelurostrongylus abstrusus

Examination for A. abstrusus was performed by conventional PCR according to Traversa and Guglielmini (2008) with minor modifications. The ITS-2 region of ribosomal DNA (~233 base pairs) was amplified using the primers listed in Table 2. PCR amplifications were carried out in a final volume of 50 μL containing: 100 pmol of each primer, 25.0 μL RED Taq ReadyMix (Sigma-Aldrich, Chemie, GmbH), 5.0 μl template DNA, and dH 2 O. Amplifications were performed using a Biometra T3 Thermocycler using the following conditions: 94 ◦ C for 7 min followed by 40 cycles of 94 ◦ C for 45 s, 50 ◦ C for 45 s, and 72 ◦ C for 45 s, and a final extension for 10 min at 72 ◦ C. DNA from 15 A. abstrusus L1

obtained from a Danish domestic cat was used as a positive control.

Amplicons were electrophoresed in a 2% agarose E-Gel™ (Invitrogen, Thermo Fisher Scientific, Denmark) and lengths were sized by comparison with a 100-bp DNA Ladder (New England BioLabsInc., Ipswich, England).