title: Development of droplet digital PCR for the detection of Babesia microti and Babesia duncani.
abstract: Babesia spp. are obligate protozoan parasites of red blood cells. Transmission to humans occurs through bites from infected ticks or blood transfusion. Infections with B. microti account for the majority of the reported cases of human babesiosis in the USA. A lower incidence is caused by the more recently described species B. duncani. The current gold standard for detection of Babesia is microscopic examination of blood smears. Recent PCR-based assays, including real-time PCR, have been developed for B. microti. On the other hand, molecular assays that detect and distinguish between B. microti and B. duncani infections are lacking. Closely related species of Babesia can be differentiated due to sequence variation within the internal transcribed spacer (ITS) regions of nuclear ribosomal RNAs. In the present study, we targeted the ITS regions of B. microti and B. duncani to develop sensitive and species-specific droplet digital PCR (ddPCR) assays. The assays were shown to discriminate B. microti from B. duncani and resulted in limits of detection of ~10 gene copies. Moreover, ddPCR for these species were useful in DNA extracted from blood of experimentally infected hamsters, detecting infections of low parasitemia that were negative by microscopic examination. In summary, we have developed sensitive and specific quantitative ddPCR assays for the detection of B. microti and B. duncani in blood. Our methods could be used as sensitive approaches to monitor the progression of parasitemia in rodent models of infection as well as serve as suitable molecular tests in blood screening.
authors.affiliation: BEI Resources, American Type Culture Collection, Manassas, VA 20110, USA.
authors.forename: Melisa
authors.initials: M
authors.lastname: Wilson
authors.affiliation: BEI Resources, American Type Culture Collection, Manassas, VA 20110, USA.
authors.forename: Kathleen C
authors.initials: KC
authors.lastname: Glaser
authors.affiliation: BEI Resources, American Type Culture Collection, Manassas, VA 20110, USA.
authors.forename: Debra
authors.initials: D
authors.lastname: Adams-Fish
authors.affiliation: BEI Resources, American Type Culture Collection, Manassas, VA 20110, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: Boley
authors.affiliation: BEI Resources, American Type Culture Collection, Manassas, VA 20110, USA.
authors.forename: Maria
authors.initials: M
authors.lastname: Mayda
authors.affiliation: BEI Resources, American Type Culture Collection, Manassas, VA 20110, USA. Electronic address: rmolestina@atcc.org.
authors.forename: Robert E
authors.initials: RE
authors.lastname: Molestina
mesh_terms: D000818:Animals; D001403:Babesia / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D041001:Babesia microti / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D006224:Cricetinae; D021901:DNA, Intergenic / Q000737:chemistry; D016054:DNA, Protozoan / Q000097:blood; D004275:DNA, Ribosomal / Q000737:chemistry; D008647:Mesocricetus; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012339:RNA, Ribosomal, 28S / Q000235:genetics; D012340:RNA, Ribosomal, 5.8S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity
chemical_list: D021901:DNA, Intergenic; D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D012337:RNA, Ribosomal, 18S; D012339:RNA, Ribosomal, 28S; D012340:RNA, Ribosomal, 5.8S
keywords: Babesia; Babesiosis; Blood infection; Detection; Digital PCR; Molecular method; Quantitation; Real time PCR