Authors

S Nak-on¹; P Campbell¹; J McIntyre¹; A Antonopoulus¹; T Chontananarth²; R Laing¹;  ¹ University of Glasgow, Glasgow, UK;  ² Srinakharinwirot University, Bangkok, Thailand

Discussion

The bovine lungworm, Dictyocaulus viviparus, is highly pathogenic and disease outbreaks can be difficult to predict and manage. High performance molecular detection tools could improve diagnosis of this parasite and help with the implementation of strategic control measures. Loop-mediated isothermal amplification (LAMP) is a rapid and isothermal DNA amplification assay, which could be developed for field-based detection. First, genomic DNA was extracted from single D. viviparus L3 larvae to amplify and clone the ITS2 DNA region into the recombinant plasmid (DviITS2). A novel DviLAMP primer set was designed to specifically target DviITS2 with the flanking 5.8S ribosomal DNA region. Genomic DNA and the DviITS2 plasmids from different individual larvae were tested as template in the LAMP protocol. Successful amplification with the LAMP primers was detected by conventional and colorimetric LAMP assays, with gel electrophoresis, real-time analysis and colour change observation. The colorimetric DviLAMP assay can specifically detect the D. viviparus ITS2 locus with naked eye observation after 45 and 90 minutes of incubation at 64 °C for 1 ng and 1 pg of the plasmid DNA, respectively. Future work will detect the dual-labelled (biotin and FAM) LAMP amplicon in a lateral flow system and investigate the utility of the assay as a point of care test to detect D. viviparus larvae in nasal swabs and/or faeces. Therefore, the development of DviLAMP could significantly improve the sensitivity of lungworm diagnosis in the field.