A novel nested-qPCR assay for detection of Babesia duncani infection in hamsters and humans

Abstract Background Babesia duncani is a causative pathogen for human babesiosis, transmitted by tick bite, blood transfusion, and pregnancy. As an important haemo protozoan disease, diagnostic assay of this pathogen is not available and its detection is neglected during transfusion and utilization of blood products. Methods In this study, primers and probes were designed in variable regions of B. duncani 18S rRNA gene. A novel molecular approach, nested quantitative polymerase chain reaction (nested-qPCR), was developed to specifically detect DNA of B. duncani in blood samples from hamster and human. Comparative analyses of our established technique with previously reported nested-PCR and microscopy were conducted using experimentally infected and clinical samples. Results The newly optimized diagnostic technique showed no cross reaction with other zoonotically important Babesia spp. such as Babesia microti, Babesia divergens, Babesia crassa, and Babesia motasi Hebei. The detection limit of nested-qPCR was approximately 1 copy of plasmid in a total of 20 µl volume or 1 infected red blood cells in 200 µl of whole blood. The specificity and sensitivity of this method are 100% and 98.6%, respectively. Comparative analyses revealed that nested-qPCR showed relatively higher efficacy and specificity for detecting B. duncani than microscopic examination and nested-PCR. Investigation of 492 specimens from patients of tick bite from Gansu Province, China presents no B. duncani infection. Conclusions In this study, we provide a novel diagnostic assay for determining B. duncani infection and investigating its prevalence in specific areas.