Development and field evaluation of a species-specific mt-COI targeted SYBR-Green Real Time PCR for detection and quantification of Haemonchus contortus in cattle in Turkey

Publication date: Available online 23 December 2019Source: Veterinary ParasitologyAuthor(s): Onder Duzlu, Alparslan Yildirim, Gamze Yetismis, Zuhal Onder, Emrah Simsek, Arif Ciloglu, Gozde Sahingoz Demirpolat, Abdullah InciAbstractHaemonchus contortus is one of the most important gastrointestinal nematodes (GINs) infecting sheep, goats, and cattle worldwide. We developed a SYBR Green real-time PCR (qPCR) assay for detection and quantification of H. contortus by using specific primers based on a conserved region of the mitochondrial cytochrome oxidase subunit I (mt-COI) gene, and evaluated this technique in the detection of H. contortus infections in cattle in Central Anatolia Region of Turkey. The newly developed qPCR assay successfully discriminated H. contortus from other GIN species infecting cattle in the specificity evaluations, with a specific melt peak of 77.5 °C. Our results revealed the efficient amplification of the proposed target COI region within the range of plasmid copies, from 2 × 106 to 2 × 101 per μl, with 96.9% efficiency, R² value of 0.999, and a slope of -3.398. Among the 920 cattle fecal samples from the field, 58 samples (6.3%) were positive with qPCR assay, whereas 45 samples (4.9%) were positive, as determined by larval culture, suggesting the utility of SYBR Green qPCR. Phylogenetic characterization of the partial COI gene of H. contortus isolates was also evaluated for 100 eggs and third stage larvae recovered from positive cattle fae...
Source: Veterinary Parasitology - Category: Veterinary Research Source Type: research