A loop-mediated isothermal amplification (LAMP) assay to identify isotype 1 β-tubulin locus SNPs in synthetic double-stranded Haemonchus contortus DNA

Abstract

Development of sustainable gastrointestinal nematode (GIN) control strategies depends on the ability to identify the frequencies of drug-susceptible and resistant genotypes in GIN populations arising from management practices undertaken on individual farms. Resistance to BZ drugs in GINs has been shown to be conferred by the presence of defined SNPs in the isotype 1 β-tubulin locus. Loop-mediated isothermal amplification (LAMP) assays are amenable to use on a range of DNA templates and are potentially adaptable to use in practical, cost-effective, pen-side diagnostic platforms that are needed to detect anthelmintic resistance in the field. In this study, we designed primers and examined LAMP assays to detect each of the three major isotype 1 β-tubulin SNPs conferring genetic susceptibility to BZ drugs. We used artificial pools of synthetic DNA, containing different proportions of susceptible and resistant SNPs to determine reproducibility of the assays. We demonstrated the detection of each of the isotype 1 β-tubulin SNPs conferring susceptibility to BZ drugs using the optimal LAMP assay. Isotype 1 β-tubulin SNP typing was effective in detecting BZ susceptibility, but the accuracy was reduced in samples with less than 60 % susceptible DNA. Our results show the potential for LAMP SNP typing to detect genetic susceptibility or resistance to anthelmintic drugs in livestock GINs, and some of the limitations in our approach that will need to be overcome in order to evaluate this assay using field samples.

Supplementary information: The online version contains supplementary material available at 10.1007/s12639-021-01414-w.

Keywords: Benzimidazole; LAMP; Nematode; Resistance; Small ruminant.

Fig. 1

Time (Ct minutes) to detect fluorescence by LAMP using different concentrations of DNA template representing susceptible and resistant Haemonchus contortus isotype 1 β-tubulin SNPs in biological replicates at codon 167 (a and b), codon 198 (c and d) and codon 200 (e and f). Fluorescence was measured each minute (considered in the machine as a cycle - Ct)

Fig. 2

Representative fluorescence of the respective LAMP assays with different concentrations of BZ susceptible Haemonchus contortus DNA with F167Y (a and b), E198A (c and d) and F200Y (e and f) SNPs. Fluorescence was measured at each minute (considered in the machine as cycle - Ct). The Ct (minutes) when the fluorescence passed the threshold was used as result. The equations were obtained from linear regression using as independent variable the concentration of susceptible DNA and dependent variable the results in Ct (min)

Fig. 3

Differences in Ct (min) amplified by LAMP between susceptible and resistant mutations in codon 167 (a), 198 (b) and 200 (c) of Haemonchus contortus, with a cut off to detect 90 % (dashed line) of susceptible. Cut off values were calculated using the linear regression described in Fig. 2