Blastocystis sp

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

2.9. Molecular detection and characterization of Blastocystis sp

We identified Blastocystis sp. by a direct PCR protocol targeting the ssu rRNA gene of the parasite ( Scicluna et al., 2006). The assay uses the pan- Blastocystis , barcode primer pair RD5/BhRDr to amplify a PCR product of ~600 bp. Amplification reactions (25 μL) included 5 μL of template DNA and 0.5 μM of each primer ( Table S1). Amplification conditions consisted of one-step of 95 ◦ C for 3 min, followed by 30 cycles of 1 min each at 94, 59 and 72 ◦ C, with an additional 2 min final extension at 72 ◦ C.

2.10. Molecular detection and characterization of Enterocytozoon bieneusi

We conducted E. bieneusi detection by a nested PCR protocol to amplify the ITS region as well as portions of the flanking large and small subunit of the ribosomal RNA gene as previously described ( Buckholt et al., 2002). We used the outer EBITS3/EBTIS4 and inner EBITS1/EBITS2.4 primer sets ( Table S1) to generate a final PCR product of 390 bp, respectively. PCR reactions (50 μL) consisted of 1 μL of template DNA and 0.2 μM of each primer. Cycling conditions for the primary PCR consisted of one step of 94 ◦ C for 3 min, followed by 35 cycles of amplification (denaturation at 94 ◦ C for 30 s, annealing at 57 ◦ C for 30 s, and elongation at 72 ◦ C for 40 s), with a final extension at 72 ◦ C for 10 min. Conditions for the secondary PCR were identical to the primary PCR except only 30 cycles were carried out with an annealing temperature of 55 ◦ C.