Development and evaluation of a quantitative PCR assay for detection of Hepatozoon sp.

With the aim to improve current molecular diagnostic techniques of Hepatozoon sp. in carnivore mammals, we developed a quantitative PCR (qPCR) assay with SYBR Green I®. The method, consisting of amplification of a 235 bp fragment of the 18S rRNA gene, is able to detect at least 0.1 fg of parasite DNA. Reproducible quantitative results were obtained over a range of 0.1 ng–0.1 fg of Hepatozoon sp. DNA. To assess the performance of the qPCR assay, DNA samples from dogs (140) and cats (50) were tested with either standard PCR or qPCR. Positive samples were always confirmed by partial sequencing of the 18S rRNA gene. Quantitative PCR was 15.8% more sensitive than standard PCR to detect H. canis in dogs. In cats, no infections were detected by standard PCR, compared to two positives by qPCR (which were infected by H. canis as shown by sequencing).