*4.4. PCR Testing*

Eight real-time PCR methods were tested on the same DNA extracts: 4 "in-house" PCRs already assessed [13–15,22] and 4 multiplex commercial PCRs: RIDA® GENE Parasitic Stool Panel II (R-Biopharm, Darmstadt, Germany), FTD® Stool parasites (Fast Track Diagnostics, Esch-sur-Alzette, Luxembourg), Amplidiag® Stool Parasites (Mobidiag, Paris, France), and Allplex® GI Parasite Assay (Seegene, Düsseldorf, Germany). The studied methods were selected based on methods used in France according to data collected by the CNR-LE for cryptosporidiosis. PCR was performed in triplicate for each tested condition on a CFX96 PCR detection system (Bio-rad, Marnes-la-Coquette, France) according to published data for "in-house" PCR and according to the manufacturer's instructions for commercial PCR (synthesized in Table 4). A total of 784 PCRs were performed (98 per tested method). In detail, each studied condition was extracted 3 times (N = 3) and run in simplicate per extract for PCR amplification. Consequently, regarding assays from range of dilution + rare species + specificity investigations respectively: 36 + 15 + 47 = 98 DNA extracts were tested for each studied method (N = 8). Assays were divided into two runs per studied method (two distinct PCR plates). A total of 16 PCR runs were done.

Results were considered positive when curves were exponential in logarithmic scale until the last cycle expected by each PCR program (Table 4).

PCR efficiencies (10\_1/slope \_1) were estimated according to Bustin et al. 2009: plotting the logarithm of the initial template concentration on the x-axis and Cq on the y-axis [35]. R<sup>2</sup> values were obtained using graphical representation on Excel software.

*Pathogens* **2021**, *10*,647


F: Forward. R: Reverse. SSU: Small subunit. COWP: *Cryptosporidium* oocyst wall protein.
