Ancylostoma duodenale qPCR

Human DNA samples, positive for A. ceylanicum, were subject to a singleplex qPCR with specific primers and probe to identify A. duodenale as previously described [43]. Reaction mixes with a final volume of 8 μL were prepared to contain 3.5 μL of GoTaq^® qPCR Master Mix (Promega), 0.71 μL of H₂O, appropriate amounts of primer and probe for A. ceylanicum (Table 1), and 3 μL of template DNA. The qPCR was run on a 5-plex Corbett RotorGene 6000 (Qiagen) with the following cycling conditions; 95°C for 15 minutes followed by 40 cycles at 95°C for 30 seconds, 55°C for 60 seconds, and 72°C for 30 seconds.

Clones of A. duodenale G-block gene fragments (Integrated DNA Technologies), used as a positive control, and nuclease-free water as negative control were run for each qPCR assay. A 1:10 dilution series of the positive control was used to prepare the standard curve.