Med. Weter. 82 (10), 459-465, 2026
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| ZOFIA NOWEK, MARCIN MICKIEWICZ, MICHAŁ CZOPOWICZ, AGATA MOROZ-FIK, ADRIAN-VALENTIN POTĂRNICHE, KINGA BIERNACKA, TOMASZ NALBERT, OLGA SZALUŚ-JORDANOW, PAWEŁ GÓRSKI, ALISTAIR ANTONOPOULOS, IWONA MARKOWSKA-DANIEL, EMILIA BAGNICKA, MARIÁN VÁRADY, JAROSŁAW KABA |
| Comparison of microchamber-based digital PCR and pyrosequencing for quantification of F200Y single nucleotide polymorphism in Haemonchus contortus larval cultures |
| Control of gastrointestinal nematode (GIN) infections in small ruminants still relies mainly on using various anthelmintic drugs, of which most popular are benzimidazoles (BZ). An inevitable consequence is development of resistance to BZ, which results from single nucleotide polymorphism (SNP) in β-tubulin isotype 1 gene. Major SNP appears to be thymine into adenine transversion at position 200 (F200Y). It can be detected using various molecular methods, of which pyrosequencing is considered the reference method owing to its proven performance in quantifying benzimidazole resistance-associated SNPs. Recently digital PCR based either on water-in-oil droplet emulsification (droplet digital PCR) or use of microchambers has been introduced. As microchamber-based digital PCR (mdPCR) has not previously been compared with pyrosequencing for quantification of the F200Y SNP in Haemonchus contortus, the aim of this study was to determine the agreement between the two methods. The study was carried out on larval cultures obtained from 63 goat herds which were confirmed to contain the third-stage larvae (L3) of H. contortus by microscopic examination and the real-time PCR. The mdPCR yielded significantly higher F200Y SNP frequency values than pyrosequencing, with a median difference of 10%. The agreement in quantitative results between both methods was only moderate (Rc = 0.52). The agreement assuming 4-category classification (0-25%, 26-50%, 51-75%, and > 75%) was moderate to good. The observed agreement was 82.5% and the chance-corrected agreement expressed using the Gwet’s AC2 unweighted coefficient was 0.789. In conclusion, the mdPCR and pyrosequencing show only moderate agreement in determination of F200Y SNP frequency in larval cultures and the former method yields significantly higher values. The higher F200Y SNP frequencies obtained by mdPCR may influence the interpretation of benzimidazole resistance levels and should therefore be considered when comparing results generated by different molecular methods. Further validation studies are needed to establish the causes of the observed differences and define the role of mdPCR in routine monitoring of benzimidazole resistance. |
| Keywords: GIN, anthelmintic resistance, digital PCR, pyrosequencing, goats |