Quantifying Cryptosporidium parvum in Feces

DNA was extracted from fecal samples using the QiaAmp PowerFecal pro DNA kit (Qiagen). qPCR was performed with the QuantStudio 3 system (Thermo Fisher Scientific) using previously described cycling conditions (24 (link)). Each sample was run as two technical replicates containing 2 μL purified fecal DNA (diluted 1:5 in low-Tris-EDTA buffer), 10 μL SYBR green QuickStart Taq ReadyMix (Sigma), and 1.6 μL of 5 μM primer solution targeting C. parvum GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (Table S2). C. parvum oocyst quantities were determined via the QuantStudio Design & Analysis New (DA2) software (Thermo Fisher Scientific) using standard curves for C. parvum gDNA. The number of oocysts per milligram of feces was calculated as the average number of C. parvum oocyst gDNA equivalents (total gDNA equivalents divided by 4) divided by the mass of the fecal sample from which DNA was purified.