The identification and semi-quantitative assessment of gastrointestinal nematodes in faecal samples using multiplex real-time PCR assays

Abstract Background The diagnosis of gastrointestinal nematode (GIN) infections in ruminants is routinely based on morphological/morphometric analysis parasite specimens recovered by coprological methods, followed larval culture (LC) techniques. Such an approach laborious, time-consuming, requires a skilled expert, and moreover suffers from certain limitations. Molecular tools are able to overcome the majority these issues, providing accurate identification species and, therefore, may be valuable sustainable control strategies. Methods Two multiplex real-time polymerase chain reaction (PCR) assays for specific detection five main one invasive GIN species, including internal amplification avoid false-negative results, were designed targeting SSU rRNA COI genetic markers, as well established ITS1/2 sequences. optimized DNA extracted directly sheep faeces verified Haemonchus contortus , Teladorsagia circumcincta Trichostrongylus colubriformis Nematodirus battus Chabertia ovina Ashworthius sidemi . Semi-quantitative evaluation infection intensity was enabled using plasmid construct dilution series with known number eggs. Assays tested 44 individually collected faecal samples three farms, results compared those egg counts (FEC) concentration McMaster technique LC. Results Multiplex PCR showed great specificity target nematodes. During samples, proved have higher sensitivity strongylid-type over FEC revealing while showing moderate agreement intensity. further clarified LC, which had confused determination spp. Conclusions Our rapid enabling simultaneous reliable semi-quantitative estimation eggs present. This increases diagnostic value add high degree precision anthelmintic efficacy, where it important identify surviving after treatment. Graphical