Method Description

Nucleic acid is extracted from feces and urine 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 a self-contained 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 18S ribosomal RNA gene of Enterocytozoon bieneusi, Encephalitozoon intestinalis, Encephalitozoon hellem, and Encephalitozoon cuniculi.

This assay utilizes 2 primers and 2 probes, which are specific for E bieneusi and 3 primers and 2 probes specific for Encephalitozoon species. 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, 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 670 emits a measurable and quantifiable light signal at a specific wavelength when E bieneusi is present, and the LC-Red 610 emits a measurable and quantifiable light signal in the presence of Encephalitozoon species.(Bernard PS, Reiser A, Pritham GH: Mutation detection by fluorescent hybridization probe melting curves. In: Meuer S, Wittwer C, Nakagawara K, eds. Rapid Cycle Real-Time PCR: Methods and Applications. Springer; 2012:11-20)