Cryptosporidium 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

2.6. Molecular detection of Cryptosporidium spp

We assessed the presence of Cryptosporidium spp . using a nested-PCR protocol to amplify a 587-bp fragment of the ssu rRNA gene of the parasite ( Tiangtip and Jongwutiwes, 2002). Amplification reactions (50 μL) included 3 μL of DNA sample and 0.3 μM of the primer pairs CR-P1/CR-P 2 in the primary reaction and CR-P3/CPB-DIAGR in the secondary reaction ( Table S1). Both PCR reactions were carried out as follows: one step of 94 ◦ C for 3 min, followed by 35 cycles of 94 ◦ C for 40 s, 50 ◦ C for 40 s and 72 ◦ C for 1 min, concluding with a final extension of 72 ◦ C for 10 min.

2.7. Molecular differential detection of Entamoeba histolytica and Entamoeba dispar

We carried out detection and differential diagnosis between pathogenic E. histolytica and non-pathogenic E. dispar by a qPCR method targeting a 172-bp fragment of the gene codifying the ssu rRNA gene of the E. histolytica / E. dispar complex ( Verweij et al., 2003a; Guti´errez-- Cisneros et al., 2010). Amplification reactions (25 μL) consisted of 3 μL template DNA, 0.5 μM of the primer set Ehd-239F/Ehd-88R, 0.2 μM of each TaqMan® probe ( Table S1), and TaqMan® Gene Expression Master Mix (Applied Biosystems, CA, USA). Detection of parasitic DNA was performed on a Corbett Rotor GeneTM 6000 real-time PCR system (Qiagen) using an amplification protocol consisting of an initial hold step of 2 min at 55 ◦ C and 15 min at 95 ◦ C followed by 45 cycles of 15 s at 95 ◦ C and 1 min at 60 ◦ C.