Development of loop-mediated isothermal amplification (LAMP) assay for rapid diagnosis of ovine theileriosis in China

摘要

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid detection method in which the target deoxyribonucleic acid (DNA) can be efficiently amplified with high specificity and sensitivity under isothermal conditions using a set of either four or six specific primers. In this study, we have identified a conserved sequence for Theileria luwenshuni (UTRlu8) and for T. uilenbergi (UTRu6) suitable for designing a set of six primers for the simultaneous detection by LAMP of these pathogens causing theileriosis in sheep and goats in China. LAMP was performed at 63°C, and the amplified DNA was detectable within 15 min. The specificity of the reaction was confirmed through EcoRI restriction enzyme digestion analysis and sequencing. The assay was proven sensitive since specific amplification was obtained from 0.1 pg DNA of T. luwenshuni or T. uilenbergi. The LAMP assay was evaluated by testing 86 field samples in comparison to the reverse line blot method, showing a sensitivity and specificity of 66.0% and 97.4%, respectively. These results indicate that the LAMP assay is rapid and simple to run, cost effective, sensitive, and specific and has potential usefulness for application in diagnostics of and epidemiological studies on T. luwenshuni and T. uilenbergi infection of small ruminants.