Abstract

Development of a new amplification-refractory mutation system for detection of a single nucleotide polymorphism linked to drug resistance in Ancylostoma caninum

Single nucleotide polymorphisms at codons 167, 198, and 200 in the β-tubulin isotype 1 gene have been associated with benzimidazole resistance. Until now, the only mutation observed in Ancylostoma caninum was at codon 200 of this gene. However, the standardized 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, followed by sequencing. The results revealed that the developed technique has high sensitivity, specificity, and reproducibility, which allow its application in the field.

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