A novel colourimetric loop-mediated isothermal amplification (LAMP) assay for rapid field-level detection of Theileria orientalis

Theileria orientalis, the causative agent of oriental theileriosis is a globally distributed protozoan parasite affecting livestock. Rapid and accurate parasite detection is crucial for effective disease management and evaluating therapeutic interventions. The high prevalence of T. orientalis in Kerala, a south Indian state demanded the development of a sensitive field tool for specific detection. Loop-mediated isothermal amplification (LAMP) is a rapid and highly sensitive nucleic acid amplification technique conducted under isothermal conditions. In this study, a LAMP assay was developed targeting the major piroplasm surface protein (MPSP) gene of T. orientalis using colourimetric dyes (both hydroxy naphthol blue (HNB) and phenol red) for improved visual detection of amplification. This assay utilised a set of six specifically designed primers, recognizing eight distinct regions on the target gene. Both wet LAMP assays demonstrated the ability to amplify DNA at levels as low as 10⁻⁴ ng (0.1 pg), corresponding to a parasitaemia level of 0.0012 % and exhibited higher detection abilities than PCR. The assay also demonstrated high specificity, with no amplification observed for DNA template from other haemoprotozoans. Positive LAMP products were identified by a distinct colour change from violet to blue and pink to yellow for HNB and phenol red dyes, respectively. Results were confirmed using agarose gel electrophoresis, showing characteristic ladder patterns. The LAMP assays detected T. orientalis in 62.8 % of samples, outperforming PCR (60 %) and microscopy (52.8 %). With a sensitivity of 100 %, specificity of 93 %, positive predictive value of 95.54 % and negative predictive value of 100 %, the wet LAMP assay demonstrated diagnostic efficacy for T. orientalis.