Table 1.
N. americanus specific primers for the mutant plasmid constructs N. americanus specific primers for the mutant plasmid constructs for three SNP positions in the β-tubulin isotype 1 gene
Fig 1.
SmartAmp2 primer design for β-tubulin mutation (SNP) detection.
Partial sequence of the β-tubulin isotype 1 gene carrying (A) SNP 167A, (B) SNP 198C, and (C) SNP 200A as well as the sequences of primers used for the SmartAmp2 assay for the three SNP positions. The locations of SNPs indicated in bold. The boost primer (BP) was used as the discrimination primer. The folding primer (FP) has a specific sequence (CCTATATATATATAGG) at the 5’ end to allow self-annealing hairpin formation.
Fig 2.
SmartAmp2 assay optimization for N. americanus β-tubulin SNP at codon 200T/A.
(A) Wild-type (WT)-specific primer amplification of WT plasmid (red circle)and no amplification with mutant-type (MT) plasmid (blue triangle) or NC (no template) (green diamond). (B) MT-specific primer amplification of MT plasmid (blue triangle) and no amplification with WT plasmid (red circle) or NC (green diamond). Assays were run in duplicate.
Fig 3.
Evaluation of SmartAmp2 SNP detection assay in pools and individual hookworm samples of eggs or larvae.
(A) Wild-type (WT)-specific primer amplification of gDNA from egg and larval pools with complete suppression of amplification using MT-specific primers. (B) WT-specific primer amplification of gDNA from single eggs and larva with complete suppression of amplification using the MT-specific primers. Amplification with WT primers, larva(e) (red circle), egg(s) (green square), and amplification with MT primers (blue triangle), for egg(s) or larva(e).
Fig 4.
SmartAmp2 assay specificity of mutant-type (MT) allele detection in the presence of wild-type (WT) DNA.
SmartAmp2 amplification for SNPs at codon 167 (first panel), and codons 198 or 200 (second panel), using the MT-specific primers with a mixture of MT plasmid and WT plasmid, 1:1 (50%; red circle), 1:9 (10%; blue triangle), 1:99; (1%; green diamond), and 0:100 (0%; purple square). Duplicate assays.
Fig 5.
Genotyping results of N. americanus samples at codon 198 with SmartAmp2 and conventional sequencing.
(A) SmartAmp2 amplification of 198A/C polymorphs using wild-type (WT) and mutant-type (MT)-primer sets. Left, center, and right panels show assay results for homozygous WT (WT/WT), mixed (WT/MT), and homozygous MT (MT/MT) samples, respectively. (B) Conventional sequencing of β-tubulin gene from left to right, WT (A/A), mixed (A/C), and MT (C/C).
Fig 6.
Evaluation of SmartAmp2 genotyping assays in fecal samples spiked with N. americanus larvae (L3).
SmartAmp2 assay using the wild-type (WT)-primer set on DNA extracted from purified L3 (no feces) (red circle), fecal samples spiked with L3 (blue triangle) and fecal samples without L3 (purple square). Negative controls (NC; no feces, no larvae) were included in each run (green diamond).