Differential detection of ovine Theileria species using loop-mediated isothermal amplification combined with nanoparticle-based lateral flow biosensor

Abstract

Ovine theileriosis is an important tick-borne protozoan disease. It has been reported that three Theileria species are responsible for ovine theileriosis in China, which are T. luwenshuni, T. uilenbergi, and T. ovis. Here, we established three detection techniques based on loop-mediated isothermal amplification (LAMP) and nanoparticle-based lateral flow biosensor (LFB) for the infection of the three Theileria species. Three LAMP primer sets were designed targeting the nucleotide sequences of the 28S rRNA gene of T. luwenshuni, T. uilenbergi, and T. ovis. We used LAMP coupled with real-time fluorescence detection to optimize the concentrations of dNTP Mix, MgSO4, and Bst 2.0 DNA polymerase, as well as the reaction temperature of the LAMP assay, and then combined LAMP with LFB (LAMP-LFB). The entire detection assay process, including genomic DNA extraction (40 min), LAMP reaction (40 min), and LFB readout (<5 min), can be completed within 85 min. The established assays can specifically detect species of T. luwenshuni, T. uilenbergi, and T. ovis infection without cross-reaction with other Theileria, Babesia, and Anaplasma species. The detection limits of the LAMP-LFB assays for T. luwenshuni, T. uilenbergi, and T. ovis plasmid templates were 2.72 × 10² copies/μL, 2.96 × 10³ copies/μL, and 3.05 × 10¹ copies/μL, respectively. Finally, we compared the established LAMP-LFB assay with the traditional PCR assay. The results showed that the total coincidence rates were 96.67 % (T. luwenshuni), 96.67 % (T. uilenbergi), and 93.33 % (T. ovis), respectively. In general, we developed a rapid, simple, sensitive, and specific technique for differential detection of T. luwenshuni, T. uilenbergi, and T. ovis infection in small ruminants.