Method Description

Nucleic acid is extracted from EDTA whole blood using the automated MagNA Pure bead-based system (Roche Molecular Systems). The extract is then transferred to individual self-contained capillary cuvettes for amplification. The LightCycler is an automated instrument that amplifies and monitors the development of target nucleic acid (amplicon) after each cycle of polymerase chain reaction (PCR).

The DNA target for PCR assay is a gene encoding the nuclear small subunit ribosomal RNA (SS-rDNA). This assay consists of 2 forward primers, 1 reverse primer, and 2 probes that are specific for the Babesia species target DNA. The specific base pair DNA target sequence is first amplified by PCR using the target-specific primers. Amplicon is then detected during melting curve analysis using fluorescence resonance energy transfer probes, which utilizes one hybridization probe with a donor fluorophore, fluorescein, at the 3' end and a second hybridization probe with an acceptor fluorophore, LC-Red 640, at the 5' end. Fluorescence is produced when the 2 probes anneal to the target sequence in close proximity to one another. The LC-Red 640 then emits a measurable and quantifiable light signal at a specific wavelength.(Burgess MJ, Rosenbaum ER, Pritt BS, et al. Possible transfusion-transmitted Babesia divergens-like/MO-1 in an Arkansas patient. Clin Infect Dis. 2017 Jun 1;64(11):1622-165)