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Background  Canine babesiosis and anaplasmosis, caused by  Babesia canis  and  Anaplasma phagocytophilum , respectively, are significant tick-borne diseases in Baltic countries. Both diseases can be diagnosed on the basis of clinicopathological findings, by direct pathogen detection in blood smears or by indirect pathogen detection; however, because of high selectivity and specificity, molecular methods may be advantageous. The goal of this study was to develop a duplex real-time polymerase chain reaction (RT-PCR) method for the detection of  B. canis  and  A. phagocytophilum  in canine clinical samples.    Methods  Sequence-based polymorphism analysis of genes encoding  B. canis -specific merozoite surface protein Bc28.1 ( Bc28.1 ) and  A. phagocytophilum  malate dehydrogenase ( mdh ) was performed on pathogen isolates present in Latvian domestic dogs. The obtained results were used to design a species-specific duplex RT-PCR assay.    Results  The presence of three  B. canis Bc28.1  gene sequence types was revealed in canine samples with a nonuniform geographical distribution, and two types of  A. phagocytophilum mdh  genes were detected. The novel duplex RT-PCR assay provided correct classification of samples positive and negative for  B. canis  and  A. phagocytophilum . The analytical sensitivity of this assay was ten gene copies/ reaction for both pathogens.    Conclusions  A novel duplex RT-PCR molecular method was developed for the detection of  B. canis  and  A. phagocytophilum  in canine clinical samples. Sequence variability of  Bc28.1  and  mdh  genes indicated the genetic variability of  B. canis  and  A. phagocytophilum  isolates occurring in Latvian domestic dogs.    Graphic AbstractSupplementary Information  The online version contains supplementary material available at 10.1186/s13071-021-04756-9.

Development of a real-time PCR method for rapid diagnosis of canine babesiosis and anaplasmosis