Troglodytella spp

K, Pamela C., oster, Lapuente, Juan, Pizarro, Andrea, Prieto-P, Laura, erez, Ramon, erez-Tanoira, Dashti, Alejandro, Begona ˜ Bailo, Muadica, Aly S., Gonz, David, alez-Barrio, Calero-Bernal, Rafael, Ponce-Gordo, Francisco & Carmena, David, 2022, Presence and genetic diversity of enteric protists in captive and semi-captive non-human primates in coteˆd’Ivoire, Sierra Leone, and Peru, International Journal for Parasitology: Parasites and Wildlife 17, pp. 26-34 : 28

2.12. Molecular detection of Troglodytella spp

Hitherto, we aimed to detect the ciliate mutualist Troglodytella spp . by a direct PCR method targeting a 401-bp fragment of the ITS region of the rDNA (ITS1- 5.8S rDNA-ITS2) ( Vallo et al., 2012). PCR reactions (25 μL) contained 2 μL of template DNA and 0.8 μM of each primer ssu-end/LSU-start ( Table S1). Conditions of PCR for ITS amplification were initial denaturation for 2 min at 94 ◦ C, 35 cycles of 45 s at 94 ◦ C, 45 s at 50 ◦ C, and 90 s at 72 ◦ C, and terminal elongation for 5 min at 72 ◦ C.

2.13. PCR and gel electrophoresis standard procedures

We carried out all the direct, semi-nested, and nested PCR protocols described above on a 2720 Thermal Cycler (Applied Biosystems). Reaction mixes included 2.5 units of MyTAQ™ DNA polymerase (Bioline GmbH, Luckenwalde, Germany), and 5 × MyTAQ™ Reaction Buffer containing 5 mM dNTPs and 15 mM MgCl 2. The specific DNA primer and probe sequences used in the present study were detailed in Table S1. We routinely used laboratory-confirmed positive and negative DNA samples of human and animal origin for each parasitic species investigated as controls and included them in each round of PCR. We visualized PCR amplicons on 1.5–2% D5 agarose gels (Conda, Madrid, Spain) stained with Pronasafe (Conda) or Gel Red (Biotium, Fremont, California, USA) nucleic acid staining solutions. We used a 100 bp DNA ladder (Boehringer Mannheim GmbH, Baden-Wurttemberg, Germany) for the sizing of obtained amplicons.