2.5. Cryptosporidium parvum DNA Amplification

For Cryptosporidium parvum DNA amplification, our in-house PCR was used following the protocol as described in Brunet et al., 2016 [22]. Briefly, the amplification of a 258-bp DNA fragment located in the 18S ribosomal ribonucleic acid (rRNA) gene (GenBank accession n°{"type":"entrez-nucleotide","attrs":{"text":"L16996","term_id":"290015","term_text":"L16996"}}L16996; positions 80 to 337) was carried out using the forward 5′ GTT AAA CTG CRA ATG GCT 3′ (Cry80F3) and reverse 5′ CGT CAT TGC CAC GGT A 3′ (Cry337R) primers, using the hybridization probes: 5 ′CCG TCT AAA GCT GAT AGG TCA GAA ACT TGA ATG 3′ Fluorescein (anchor probe) and 5′ Red 640-GTC ACA TTA ATT GTG ATC CGT AAA G 34 Phosphate (sensor probe). Primers and probes were used at a concentration of 10 μM. Five microliters of DNA extracts were added to a final reaction volume of 20 μL to each amplification reaction tube. Thermocycling and fluorescence detection were performed on the LightCycler 2.0 Roche Molecular Systems, Inc. (Rotkreuz, Switzerland). One negative (i.e., sterile water or stool samples without Cryptosporidium oocysts and other parasites) and one positive (i.e., stool samples containing C. parvum at the concentration of 100 oocysts/mL) controls were included in each assay. All in all, a total of 625 PCRs were carried out, including 605 Cryptosporidium-specific PCRs for Cryptosporidium DNA detection in stool extracts, and 20 PCRs for the internal control detection in stool extracts.