title: Detection and species identification of microsporidial infections using SYBR Green real-time PCR.
abstract: 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.
authors.affiliation: Department of Clinical Parasitology, Hospital for Tropical Diseases, University College London Hospitals NHS Foundation Trust, Mortimer Market, Capper Street, London WC1E 6JB, UK.
authors.forename: Spencer D
authors.initials: SD
authors.lastname: Polley
authors.affiliation: Department of Clinical Parasitology, Hospital for Tropical Diseases, University College London Hospitals NHS Foundation Trust, Mortimer Market, Capper Street, London WC1E 6JB, UK.
authors.forename: Samuel
authors.initials: S
authors.lastname: Boadi
authors.affiliation: Department of Clinical Parasitology, Hospital for Tropical Diseases, University College London Hospitals NHS Foundation Trust, Mortimer Market, Capper Street, London WC1E 6JB, UK.
authors.forename: Julie
authors.initials: J
authors.lastname: Watson
authors.affiliation: Health Protection Agency, Regional Microbiology Network, North West Regional Laboratory, PO Box 209, Clinical Sciences Building, Manchester Royal Infirmary, Oxford Road, Manchester M13 9WZ, UK.
authors.forename: Alan
authors.initials: A
authors.lastname: Curry
authors.affiliation: London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK.|Department of Clinical Parasitology, Hospital for Tropical Diseases, University College London Hospitals NHS Foundation Trust, Mortimer Market, Capper Street, London WC1E 6JB, UK.
authors.forename: Peter L
authors.initials: PL
authors.lastname: Chiodini
mesh_terms: D052160:Benzothiazoles; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D003959:Diamines; D009670:Encephalitozoon cuniculi / Q000235:genetics / Q000302:isolation & purification*; D021862:Enterocytozoon / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D016881:Microsporidiosis / Q000175:diagnosis* / Q000382:microbiology*; D008969:Molecular Sequence Data; D009930:Organic Chemicals; D021864:Pleistophora / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013194:Staining and Labeling / Q000379:methods; D044366:Transition Temperature
chemical_list: D052160:Benzothiazoles; D004271:DNA, Fungal; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I