Loop mediated isothermal amplification (LAMP) of Theileria annulata DNA

In the past three decades, as an alternative to PCR (polymerase chain reaction) new diagnostic techniques like LAMP (loop mediated isothermal amplification) whereby target DNA can be amplified under isothermal conditions without using thermocycler have been developed. The LAMP method allows the synthesis of large amounts of DNA in a short time with high specificity and rapid and easy detection of generated products. In this study, specificity and sensitivity of LAMP method was evaluated for the detection of T. annulata in acute infected and/or carriers cattle using primer pair specifically designed to amplify merozoite surface antigen gene (Mero1), 30 kDa major merozoite surface antigen gene (Tams-1) and cytochrome b gene of T. annulata. Primer pairs with highest sensitivity were used to evaluate the applicability of LAMP to the field samples. Two LAMP primers (CYTOB1 and CYTOB341) targeting cytochrome b gene specifically amplified DNA of different T. annulata isolates successfully while no amplification was seen in other species DNAs and BL20. CYTOB1 primers detected T. annulata Ankara/D7 DNA up to 2 fg, however the detection limit of CYTOB341 was 10 fold lower. The sensitivity of CYTOB1 LAMP assay was same with F3/B3 PCR, however when compared with that of cytob1 PCR a 10 fold lower sensitivity was found. The LAMP product was confirmed by restriction digestion and sequencing. Results obtained from this study indicated that none of the designed primer pairs specific to target genes (Tams-1 and Mero1), except cytochrome b gene was able to specifically and sensitively detect different isolates of T. annulata. Consequently, it was shown that LAMP method using CYTOB1 primers is less effective than the cytob1 PCR in terms of detecting T. annulata in the field samples.