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

• Published in: Experimental parasitology Vol. 125; no. 3; pp. 196 - 201
• Main Authors: Njiru, Zablon K., Ouma, Johnson O., Enyaru, John C., Dargantes, Alan P.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier Inc 01.07.2010; Elsevier
• Subjects: Animals; Biological and medical sciences; Camelus; Diagnosis; DNA Primers - chemistry; DNA, Protozoan - chemistry; Enzymes; Fundamental and applied biological sciences. Psychology; Humans; Lateral Flow Dipstick; Life cycle. Host-agent relationship. Pathogenesis; Loop-mediated Isothermal Amplification (LAMP); Nucleic Acid Amplification Techniques - methods; Polymerase Chain Reaction; Protozoa; Sensitivity and Specificity; Surra; Trypanosoma - classification; Trypanosoma - genetics; Trypanosoma - isolation & purification; Trypanosoma evansi; Trypanosoma evansi type B; Loop-mediated Isothermal Amplification (LAMP); Trypanosoma evansi type B; Diagnosis; Lateral Flow Dipstick; Surra; Kinetoplastida; Amplification; Trypanosoma evansi; Protozoa; Diagnosis (taxonomy); Parasite; Detection
• ISSN: 0014-4894; 1090-2449
• DOI: 10.1016/j.exppara.2010.01.017

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 20–25min at 63°C using a real time PCR machine. Restriction enzyme AluI digestion of the amplicon gave the predicted 83bp and 89bp sized bands and the LAMP product melt curves showed consistent melting temperature (Tm) of ∼89°C. The assay analytical sensitivity is ∼0.1tryps/ml while that of classical PCR test targeting the same gene is ∼10tryps/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.