Detection and Differentiation of Babesia microti and Other Pathogenic Babesia Species by a Single-Amplicon, Dual-Probe Real-Time PCR Assay
Methods: A 190bp segment of the 18S rRNA gene (18S) was amplified with a common set of PCR primers and detected by two different probes: (1) Babesia microti, (2) other Babesia spp. of human relevance. Test performance was established with 39 whole blood specimens from patients with known B. microti infections diagnosed by microscopy (n=8) and PCR (n=31). Whole blood was also spiked with B. duncani organism (ATCC PRA-302) and plasmids covering the 18S target of MO-1, B. divergens, and EU-1.
Results: Analytical sensitivity for both channels was between 285-435 copies/mL (8-13 copies/reaction). All positive whole blood samples produced the expected results. Positive patient specimens had a mean of 2.6E+08 copies/mL (range 2.5E+03 to 1.2E+10). No cross-reactivity was observed with 56 other human pathogens, including the 5 relevant Plasmodium spp., Trypanosoma cruzi, and Leishmania infantum.
Conclusion: The incidence of babesiosis has increased in recent years and previously unrecognized species have been shown to cause human infections. We describe the first multiplex, real-time PCR assay for sensitive detection of all Babesia species of known human relevance using an innovative single-amplicon, dual-probe design. Given the highly conserved 18S target, this assay will enable us to identify and monitor circulation of unusual Babesia species by subsequent 18S sequencing.
Gen-Probe: Collaborator, Kits supplied for study
C. Ginocchio, None
R. Schlaberg, Epoch Biosciences: Collaborator, Research reagents
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