The loop-mediated isothermal amplification (LAMP) reaction is a method that amplifies with high sensitivity, efficiency, and rapidity,
deoxyribonucleic acid (
DNA) under isothermal condition in simple
incubators. Two primer sets for the LAMP method were designed using the nucleotide sequences of
18S rRNA gene of Babesia sp. BQ1 (Lintan) and Babesia sp. Xinjiang-2005 isolated in China. The primers were used to detect parasite
DNA extracted from infected blood and purified parasites by LAMP. The specific ladder bands were amplified from the autologous genomic
DNA of two Babesia species, respectively, and did not cross-react with the genomic
DNA of Theileria sp. China 1, Theileria sp. China 2, B. bovis, Theileria sp. (Japan) and sheep. The LAMP was sensitive enough to detect 0.02 pg and 0.2 pg genomic
DNA of Babesia sp. BQ1 (Lintan) and Babesia sp. Xinjiang-2005, respectively, from 10-fold serially diluted samples corresponding to the amount of
DNA present in 50 microl of 0.000002% and 0.00002% parasitemic erythrocytes. Furthermore,
DNA extracted from blood of intact (non-splenectomized) sheep experimentally infected with Babesia sp. BQ1 (Lintan) and Babesia sp. Xinjiang-2005 was amplified by the LAMP from week 1 to 9 and week 2 and 3 post-
infection, respectively, demonstrating the high sensitivity of these primers. Of 365 samples collected from Gansu province, 14.3% (52/365) were positively detected by the LAMP. Of 145 samples collected on filter papers (Whatman) from the grazing sheep in Xinjiang province, 3.5% (5/145) were positive. These results show that the LAMP could be an alternative diagnostic tool for the detection of babesial
infection in sheep and goats.