Cross-Genera PCR Amplification of DNA from Apicomplexan Parasites

Abstract

Background: The discovery of an unexpected genetic sequence raised doubts about the specificity of a primer pair targeting Babesia spp. and Theileria spp. This study aimed to check the specificity of this primer pair. Methods: Conventional end-point PCR and real-time PCR protocols using primers targeting the 18S rRNA gene V4 hypervariable region of Babesia spp. and Theileria spp. were tested for potential cross-genera amplification using DNA from a palette of parasitic protists and pathogenic bacteria as a template. These investigations took place at the Ludwig Maximilian University of Munich (Germany) in 2010 as part of the EDEN project. Results: Successful amplification was obtained with DNA from five apicomplexan genera: Babesia, Theileria, Hepa­tozoon, Toxoplasma, and Hammondia. No amplicons were obtained when DNA from Leishmania infantum or bacte­ria within the genera Borrelia, Leptospira or Anaplasma was used as a template. Conclusion: This cross-genera amplification ability is useful for the quick exclusion of many parasite species from PCR negative diagnostic samples. Accurate species identification from PCR positive samples requires genetic se­quencing of the amplicon.

Keywords

• Babesia
• Theileria
• Hepatozoon
• Toxoplasma
• Hammondia