Preliminary results of the recombinase polymerase amplification technique for the detection of Haemonchus contortus from Hungarian field samples

• Published in: Veterinary parasitology Vol. 320; p. 109974
• Main Authors: Khangembam, Rojesh, Vass, Nóra, Morrison, Alison, Melville, Lynsey A., Antonopoulos, Alistair, Czeglédi, Levente
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.08.2023
• Subjects: Animals; Diagnostic; Haemonchiasis - diagnosis; Haemonchiasis - parasitology; Haemonchiasis - veterinary; Haemonchus; Haemonchus - genetics; Hungary; Isothermal amplification; Point of care; Recombinases; RPA; Ruminants; Hungary; Haemonchus; Diagnostic; RPA; Isothermal amplification; Point of care
• ISSN: 0304-4017; 1873-2550
• DOI: 10.1016/j.vetpar.2023.109974

Haemonchus contortus is a parasitic nematode of small ruminants responsible for significant economic losses and animal health concerns globally. Detection of gastrointestinal nematode (GIN) infection in veterinary practice typically relies on microscopy-based methods such as the faecal egg count and morphological identification of larval culture. However, mixed co-infections are common and species-specific identification is typically time-consuming and expertise-intensive. Compounded by increasing anthelmintic resistance, there is an urgent need to implement the molecular diagnosis of GIN in the livestock industry, preferably in field settings. Advances in isothermal amplification techniques including recombinase polymerase amplification (RPA) assays could improve this. Yet, constraints in RPA kit availability and amplicon detection systems limit the use of this technology in point of care settings. In this study, we present an early-stage, proof-of-concept demonstration of RPA targeting the internal transcribed spacer (ITS2) region of H. contortus. Having tested against eight closely related nematodes and also against five farm isolates in Eastern Hungary, preliminary results derived from a comparative analysis of 3 primer sets showed the assay detects H. contortus DNA and has a limit of detection of 10−5 ng/μl. We also tested an end-result naked eye detection system using various DNA binding dyes, of which EvaGreen® dye was successful for a qualitative RPA detection that could be adaptable at farm sites. [Display omitted] •Molecular-based diagnosis of haemonchosis is difficult to adopt on farm sites.•Isothermal amplification-based techniques such as RPA could address this bottleneck.•RPA end-result detection system is limited with the currently available kits.•Our qualitative proof of concept study reports a colourimetric result detection system.