Development and evaluation of a loop-mediated isothermal amplification method for rapid detection of Anaplasma ovis.

Anaplasma ovis is an intraerythrocytic rickettsial pathogen of small ruminants. Loop-mediated isothermal amplification (LAMP) is a nucleic acid detection method in which the target DNA can be efficiently amplified with high specificity and sensitivity under isothermal conditions. In this study, a LAMP method was developed for the specific detection of A. ovis, using LAMP primers designed on the basis of the major surface protein 4 gene. LAMP was performed at 65 °C for 30 min. Its specificity was confirmed by successful amplification of several A. ovis isolates and through EcoRI restriction analysis of LAMP products. No cross-reactivity with the A. marginale Lushi isolate, Mycoplasma mycoides subsp. capri, Chlamydophila psittaci, Theileria ovis, T. luwenshuni, T. uilenbergi, or the Babesia sp. Xinjiang isolate was observed. Detection using the LAMP method was compared with that using conventional PCR in 227 field samples; LAMP demonstrated a sensitivity of 95.45%. In summary, LAMP is a specific, sensitive, and rapid test for the diagnosis of A. ovis infection, with the potential to be standardized as a detection method for A. ovis in areas of endemicity.