Pcr grade water

A sequence of the cytb gene from GenBank (KC305876.1) [16 (link)] was used for the development of novel primers for this locus. Hence, primers Slcytb-F (5′-ACT GCG GGG GAG TCT TTC T-3′) and Slcytb-R (5′-AGT AAT AAC AAC CGC CGC CC-3′) were designed to amplify a 260 bp fragment of the cytb gene. Primers were used at a final concentration of 250 nM. Each reaction consisted of 0.5 μl of each primer, 0.6 μl of SYTO-9 (Invitrogen), 5.4 μl of PCR grade water (Biological Industries Inc.), 10 μl of Maxima HotStart Master Mix® (Thermo Fisher Scientific Inc.) and 3 μl of DNA. The program consisted of an initial hold at 95 °C for 4 min and 45 cycles of 95 °C for 5 s, 57 °C for 15 s and 72 °C for 5 s. A melt curve from 75 °C to 90 °C was constructed, with 1 °C/s increments, followed by a hybridization step. The HRM curve was constructed from 75 °C to 90 °C with 0.1 °C/s increments.
All HRM qPCRs were run in the Rotor Gene 6000 Cycler (Qiagen, Hilden, Germany). Each reaction was run with positive, negative and non-template controls (NTC). Reaction controls included S. lupi adult-DNA (positive control), DNA from a S. lupi-negative dog fecal sample (negative control, determined by fecal flotation in three consecutive samples), a point of the standard curve by triplicate and NTC with PCR-grade water.

The S. lupi ITS1 spacer was characterized from four adult S. lupi specimens using primers rDNA2 [13 ] and rDNA1.58S [14 ], following a previously described protocol [14 ]. Then, the amplified sequence was inserted into pCR2-TOPO vectors (Thermo Fisher Scientific Inc.) and cloned. Specific primers were designed using Primer-BLAST [15 (link)], to amplify a 135 bp fragment within the ITS1 of S. lupi. The HRM qPCR assay used primers SlITS-F (5′-AAA CGG TGT CCC ATG TTG G-3′) and SlITS-R (5′-GCA GCA CAA TAG CTT GAC GC-3′) at a final concentration of 500 nM. Each reaction consisted of 1 μl of each primer, 0.6 μl of SYTO-9 (Invitrogen, California, USA), 4.4 μl of PCR grade water (Biological Industries Inc.), 10 μl of Maxima HotStart Master Mix® (Thermo Fisher Scientific Inc.) and 3 μl of DNA. The amplification reaction consisted of an initial hold at 95 °C for 4 min, followed by 45 cycles of 95 °C for 5 s, 60 °C for 15 s and 72 °C for 10 s. A melt curve from 80 °C to 95 °C was constructed with increments of 1 °C/s, followed by a hybridization step from 90 °C to 50 °C. An HRM curve was constructed from 70 °C to 85 °C, with 0.1 °C/s increments.

DNA was extracted from 0.2 g of fecal sample using the Qiagen Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany) with some modifications [34 (link)]. DNA purity and concentration were verified using a NanoDrop spectrophotometer (Thermo-Fisher Scientific, Waltham, MA, USA).
After DNA extraction, all samples were tested in triplicates by quantitative PCR, coupled with a high-resolution melt analysis (HRM qPCR) that detects a 135 bp fragment of the internal transcribed spacer 1 (ITS1) of S. lupi. Primers SlITS1-F and SlITS1-R were used at final concentrations of 500 mM, and the PCR program was run as previously described [34 (link)]. Each PCR run included DNA from a S. lupi eggs suspension as positive control, DNA from a S. lupi-negative fecal sample as negative control (determined negative by fecal flotation of three consecutive fecal samples) and a non-template control with PCR-grade water (Biological Industries, Beit-Haemek, Israel). Samples were considered positive when all three replicates had been amplified.
Amplicons from positive DNA samples were purified (Exo-SAP, New England Bio-Labs, Ipswich, MA, USA) and sequenced using the BigDye Terminator cycle sequencing chemistry (Applied Biosystems ABI3700 DNA Analyzer and ABI’s Data collection and Sequence analysis software, ABI, Carlsbad, CA, USA).