title: Species-specific detection of three human-pathogenic microsporidial species from the genus Encephalitozoon via fluorogenic 5' nuclease PCR assays.
abstract: This study describes fluorogenic 5' nuclease PCR assays suitable for rapid, sensitive, high-throughput detection of the human-pathogenic microsporidial species Encephalitozoon hellem, E. cuniculi and E.intestinalis. The assays utilize species-specific primer sets and a genus-specific dual fluorescent-labeled probe that anneals to a region within the Encephalitozoon 16S rRNA gene. The assay design theoretically permits the probe to be used either with one set of primers for species-level determination or with a combination of all three primer sets for a genus-level screening of samples. The linear range of all three species-specific calibration curves that were developed using serial ten-fold dilutions of genomic DNA isolated from hemacytometer counted spores was determined to be between 10(4) and 10(-1) spores per PCR sample. The coefficients of variation were < or =5.2% over the entire 5-log span of each calibration curve. When DNA isolated from flow cytometric enumerated spores from each of the three Encephalitozoon species was used to evaluate the quantitative capability of the species' respective calibration curves, the results from 34 out of 36 (94%) samples were within 2 standard deviations. The species-specificity of each assay was confirmed using DNA isolated from 10(4) spores from each of the other two Encephalitozoon species as well as DNA extracted from numerous other protozoa, algae and bacteria.
authors.affiliation: National Exposure Research Laboratory, United States Environmental Protection Agency, Cincinnati, OH 45268, USA.
authors.forename: J D
authors.initials: JD
authors.lastname: Hester
authors.forename: M
authors.initials: M
authors.lastname: Varma
authors.forename: A M
authors.initials: AM
authors.lastname: Bobst
authors.forename: M W
authors.initials: MW
authors.lastname: Ware
authors.forename: H D A
authors.initials: HD
authors.lastname: Lindquist
authors.forename: F W
authors.initials: FW
authors.lastname: Schaefer
mesh_terms: D000818:Animals; D002138:Calibration; D017931:DNA Primers; D016054:DNA, Protozoan / Q000032:analysis; D003851:Deoxyribonucleases; D003937:Diagnosis, Differential; D016819:Encephalitozoon / Q000235:genetics / Q000302:isolation & purification*; D016890:Encephalitozoonosis / Q000175:diagnosis*; D005456:Fluorescent Dyes; D006801:Humans; D016814:Microsporidia / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D016054:DNA, Protozoan; D005456:Fluorescent Dyes; D012336:RNA, Ribosomal, 16S; D003851:Deoxyribonucleases