title: Loop-mediated Isothermal Amplification (LAMP) test for detection of Trypanosoma evansi strain B.
abstract: 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 degrees 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 (T(m)) of approximately 89 degrees C. The assay analytical sensitivity is approximately 0.1tryps/ml while that of classical PCR test targeting the same gene is approximately 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.
authors.affiliation: Division of Health Sciences, School of Nursing and Midwifery, Murdoch University, Education Drive, Mandurah, WA, Australia. Z.njiru@murdoch.edu.au
authors.forename: Zablon K
authors.initials: ZK
authors.lastname: Njiru
authors.forename: Johnson O
authors.initials: JO
authors.lastname: Ouma
authors.forename: John C
authors.initials: JC
authors.lastname: Enyaru
authors.forename: Alan P
authors.initials: AP
authors.lastname: Dargantes
mesh_terms: D000818:Animals; D002162:Camelus; D017931:DNA Primers / Q000737:chemistry; D016054:DNA, Protozoan / Q000737:chemistry; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014345:Trypanosoma / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D017931:DNA Primers; D016054:DNA, Protozoan