Method Description

Nucleic acid is extracted from clinical specimens using the automated MagNA Pure LC instrument system. The extract is then transferred to individual wells of a 96-well plate 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 the 283-base pairs plasminogen-binding protein gene (OppA2), which is present at a frequency of 1 copy per organism in all 4 confirmed pathogenic species of the Borrelia burgdorferi sensu lato genogroup (B burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, and Borrelia mayonii). A specific base pair DNA target sequence is amplified by PCR. The detection of amplicon is based on fluorescence resonance energy transfer, which utilizes 1 hybridization probe with a donor fluorophore, fluorescein, at the 3' end, and a second hybridization probe with an acceptor fluorophore, LC-Red 610, at the 5' end. When the target amplicon is present, the LC-Red 610 emits a measurable and quantifiable light signal at a specific wavelength. Presence of the specific organism nucleic acid may be confirmed by performing a melting curve analysis of the amplicon. Using features of the melting curve analysis, the assay primers and specific hybridization probes are able to detect and differentiate B burgdorferi sensu stricto from B mayonii, B afzelii, and B garinii, although the melting curve analysis cannot differentiate between B afzelii and B garinii. Each assay run can be completed within 60 minutes.(Unpublished Mayo method)