Table 1.
Filarial nematodes used to assess the analytical specificity of the qPCR assay.
Table 2.
GenBank accession numbers (AN) of mitochondrial cytochrome c oxidase subunit 1 sequences of filarial nematodes used for primers and TaqMan-probe design.
Table 3.
Skin samples tested for Onchocerca lupi by qPCR, divided (Groups 1–5) according to the parasitic load (mfs) microscopically detected.
The mean, minimum, maximum and standard deviation (sd) values of the threshold cycle (Cq), parasite load (Starting Quantity (SQ) value, expressed as ng/μl of DNA for reaction) and microfilariae concentration, assessed by qPCR is reported.
Table 4.
Blackflies and mosquitoes/midges specimens used to test the analytical specificity of qPCR assay.
Fig 1.
Assessment of the specificity of qPCR assay in the detection of Onchocerca lupi DNA.
The amplification plot is represented by the fluorescent signal accordingly to relative fluorescence units (RFU) and threshold cycle.
Fig 2.
Standard curves generated from serial dilutions of (A) genomic DNA from adult (from 8 × 104 to 8 × 10−3 fg/2μl of reaction) and microfilariae (B) (from 3.6 ×10−1 ng/2μl to 3.6 ×101 fg/2μl of reaction) of Onchocerca lupi. Each point was tested in triplicate. Slope, efficacy and R2 are reported on the bottom.
Fig 3.
Detection limit of the conventional PCR assay determined by 10-fold serial dilution of genomic DNA of microfilariae and adult of Onchocerca lupi.
Lanes 1–4, from 3.6 ×101 pg/2μl to 3.6 ×10−3 pg/2μl of O. lupi mfs DNA (i.e., from 1 to 1×10−4 mfs); Lanes 5–15, from 8 ×101 ng/2μl to 8 x 10−3 fg/2μl of O. lupi adult DNA; Line 16, no-DNA control; M, 100 bp DNA marker.