Development of a Loop-Mediated Isothermal Amplification (LAMP) Assay Targeting the Citrate Synthase Gene for Detection of Ehrlichia canis in Dogs

Abstract

Canine monocytic ehrlichiosis caused by Ehrlichia canis is one of the leading tick-borne diseases of dogs, particularly in tropical countries. A highly sensitive and specific diagnostic method is essential for early detection to facilitate treatment. This study was conducted to develop E. canis loop-mediated isothermal amplification (LAMP) assay, a highly sensitive yet simple molecular technique, targeting the citrate synthase (gltA) gene of E. canis. Canine blood samples were subjected to conventional PCR targeting E. canis gltA. After analysis of the sequences of PCR amplicons, LAMP primers were generated. The optimum temperature and time for the LAMP assay were determined using eight samples—after which, the effectiveness and reproducibility of LAMP were verified by testing 40 samples, which included PCR-positive and negative samples. The detection limit was also established. The optimal condition for the assay was 61 °C for 60 min. Compared to PCR, the LAMP assay had a relative sensitivity and specificity of 92.5 and 100%, respectively. Statistical analysis using McNemar’s test showed that the E. canis LAMP assay has no significant difference with PCR. Therefore, the LAMP assay developed in this study may be used as an alternative to PCR in the detection of E. canis.