Loop-mediated Isothermal Amplification (LAMP) test for detection of Trypanosoma evansi strain B

Camel Trypanosomiasis (Surra) is mainly caused by Trypanosoma evansi strains that express variable surface glycoprotein (VSG) RoTat 1.2. However, in Kenya a second causative strain that does not express RoTat 1.2 VSG (T. evansi type B) has been identified. The prevalence of T. evansi type B largely remains unknown due to inadequate diagnostic assay. This work reports the development of a sensitive and specific diagnostic assay capable of detecting T. evansi type B based on the strategy of Loop-mediated Isothermal Amplification (LAMP) of DNA. The test is rapid and amplification is achieved within 20-25 min at 63 degrees C using a real time PCR machine. Restriction enzyme Alul digestion of the amplicon gave the predicted 83 bp and 89 bp sized bands and the LAMP product melt curves showed consistent melting temperature (T-m) of similar to 89 degrees C. The assay analytical sensitivity is similar to 0.1 tryps/ml while that of classical PCR test targeting the same gene is similar to 10 tryps/ml. There was a 100% agreement in detection of the LAMP amplification product in real time, gel electrophoresis, on addition of SYBR Green I, and when using chromatographic Lateral Flow Dipstick (LFD) format. The use of the LAMP test revealed nine more T. evansi type B DNA samples that were not initially detected through PCR. The robustness and higher sensitivity of the T. evansi type B LAMP assay coupled with the visual detection of the amplification product indicate that the technique has strong potential as a point-of-use test in surra endemic areas. (C) 2010 Elsevier Inc. All rights reserved.