Detection and species identification of microsporidial infections using SYBR Green real-time PCR

Diagnosis of microsporidial infections is routinely performed by light microscopy, with unequivocal non-molecular species identification achievable only through electron microscopy. This study describes a single SYBR Green real-time PCR assay for the simultaneous detection and species identification of such infections. This assay was highly sensitive, routinely detecting infections containing 400 parasites (g stool sample)(-1), whilst species identification was achieved by differential melt curves on a Corbett Life Science Rotor-Gene 3000. A modification of the QIAamp DNA tissue extraction protocol allowed the semi-automated extraction of DNA from stools for the routine diagnosis of microsporidial infection by real-time PCR. Of 168 stool samples routinely analysed for microsporidian spores, only five were positive by microscopy. By comparison, 17 were positive for microsporidial DNA by real-time analysis, comprising 14 Enterocytozoon bieneusi, one Encephalitozoon cuniculi and two separate Pleistophora species infections.