Loop-mediated isothermal amplification assay for the detection of Ehrlichia canis DNA in blood samples from dogs

La bacteria rickettsia Ehrlichia canis es el agente etiologico de la ehrlichiosis monocitica canina, una de las mas importantes enfermedades caninas transmitidas por garrapatas. En este estudio, un ensayo de amplificacion isotermica de ADN mediada por asas (LAMP) se desarrollo para la deteccion del ADN de E. canis usando iniciadores LAMP enfocando el operon groESL. Se produjeron reacciones a 60°C por 60 min y los resultados se visualizaron por electroforesis en gel. Se obtuvo una exitosa amplificacion al usar ADN plasmidico conteniendo un fragmento del operon groESL y ADN extraido de muestras de sangre que dieron positivo para E. canis via PCR en tiempo real. La especificidad de amplificacion fue confirmada por la enzima de restriccion EcoRI de sitios internos en iniciadores LAMP y no se observo ninguna reactividad cruzada con muestras de sangre positivas para Babesia spp., otro agente patogeno comun transmitido por garrapatas. El alto costo de analisis de acido nucleico es uno de los inconvenientes para su uso a gran escala como pruebas diagnosticas rutinarias. El ensayo LAMP para E. canis desarrollado aqui es una alternativa interesante para PCR, una vez que no requiere un termociclador programado y, consecuentemente, reduce costos para el laboratorio clinico veterinario.

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