title: Development of a new amplification-refractory mutation system for detection of a single nucleotide polymorphism linked to drug resistance in Ancylostoma caninum.
abstract: Single nucleotide polymorphisms at codons 167, 198, and 200 in the β-tubulin isotype 1 gene have been associated with benz-imidazole resistance. Until now, the only mutation observed in Ancy-lostoma caninum was at codon 200 of this gene. However, the standard-ized methodologies used to detect mutations in this species are faulty. The objective of this study was to standardize a molecular technique based on amplification-refractory mutation system-polymerase chain reaction (ARMS-PCR) for detecting the mutation at codon 200 in the A. caninum β-tubulin isotype 1 gene. Controls were synthesized both for the absence of the mutation, using conventional PCR, and for the presence of the mutation, using the Megaprimer-PCR technique. After standardization of the ARMS-PCR using the controls, the technique was validated through an analysis of 75 A. caninum DNA samples, fol-lowed by sequencing. The results revealed that the developed technique has high sensitivity, specificity, and reproducibility, which allow its ap-plication in the field.
authors.affiliation: Departamento de Parasitologia, Universidade Federal de Minas Gerais, Instituto de Ciências Biológicas, Belo Horizonte, MG, Brasil.
authors.forename: L F
authors.initials: LF
authors.lastname: Furtado
authors.affiliation: Departamento de Parasitologia, Universidade Federal de Minas Gerais, Instituto de Ciências Biológicas, Belo Horizonte, MG, Brasil elidam.rabelo@gmail.com.
authors.forename: É M
authors.initials: ÉM
authors.lastname: Rabelo
mesh_terms: D000722:Ancylostoma / Q000187:drug effects / Q000235:genetics*; D000818:Animals; D000871:Anthelmintics / Q000494:pharmacology; D001562:Benzimidazoles / Q000494:pharmacology; D003062:Codon; D004351:Drug Resistance / Q000235:genetics*; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D014404:Tubulin / Q000235:genetics*
chemical_list: D000871:Anthelmintics; D001562:Benzimidazoles; D003062:Codon; D014404:Tubulin; C031000:benzimidazole