Evaluation of a Loop-Mediated Isothermal Amplification Technique for the Rapid Visual Detection of Hepatozoon canis Infection

BACKGROUND: Laboratory diagnosis of Hepatozoon canis infection is tedious, especially in chronic and/or latent infections. PURPOSE: The study was planned to develop a simple read out loop mediated isothermal amplification (LAMP) assay targeting a partial 18S rRNA gene of H. canis with naked eye visualisation of LAMP products. METHODS: A LAMP assay was employed to assess the DNA amplification by adding SYBR Green I dye for naked eye inspection of DNA accumulating in reaction tubes. Positive amplification was read through observation of change in colour of reaction mixture following addition of dye. The visual results were further verified with those of agarose gel electrophoresis. Genomic DNA of other haemoparasites of dog viz. Babesia vogeli, B. gibsoni, Ehrlichia canis and Trypanosoma evansi along with no-template control were used to determine the specificity of assay. RESULTS: Among the 109 blood samples presented at Small Animal Clinics, Teaching Veterinary Clinical Complex, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab (India) tested, 39 revealed colour change from orange to green indicating positive reaction while 70 were negative as revealed by no colour change. The results of visual inspection were comparable to those obtained by agarose gel electrophoresis. The LAMP primers specifically amplified H. canis DNA, whereas no amplification was detected in DNA samples of other haemoparasites and no-template control revealing specificity of the assay. The diagnostic sensitivity and specificity (95% CI) of visual LAMP assay with respect to microscopy in detection of H. canis varied from 100% (15.81–100.00%) and 65.42% (55.61–74.35%), respectively. CONCLUSION: The present investigation has developed a specific and rapid LAMP assay for the detection of H. canis, using SYBR Green I dye, which has practical applications for the screening of field samples.