title: Development and Evaluation of Three Real-Time PCR Assays for Genotyping and Source Tracking Cryptosporidium spp. in Water.
abstract: The occurrence of Cryptosporidium oocysts in drinking source water can present a serious public health risk. To rapidly and effectively assess the source and human-infective potential of Cryptosporidium oocysts in water, sensitive detection and correct identification of oocysts to the species level (genotyping) are essential. In this study, we developed three real-time PCR genotyping assays, two targeting the small-subunit (SSU) rRNA gene (18S-LC1 and 18S-LC2 assays) and one targeting the 90-kDa heat shock protein (hsp90) gene (hsp90 assay), and evaluated the sensitivity and Cryptosporidium species detection range of these assays. Using fluorescence resonance energy transfer probes and melt curve analysis, the 18S-LC1 and hsp90 assays could differentiate common human-pathogenic species (C. parvum, C. hominis, and C. meleagridis), while the 18S-LC2 assay was able to differentiate nonpathogenic species (such as C. andersoni) from human-pathogenic ones commonly found in source water. In sensitivity evaluations, the 18S-LC2 and hsp90 genotyping assays could detect as few as 1 Cryptosporidium oocyst per sample. Thus, the 18S-LC2 and hsp90 genotyping assays might be used in environmental monitoring, whereas the 18S-LC1 genotyping assay could be useful for genotyping Cryptosporidium spp. in clinical specimens or wastewater samples.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, School of Resources and Environmental Engineering, East China University of Science and Technology, Shanghai, China Division of Foodborne, Waterborne and Environmental Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
authors.forename: Na
authors.initials: N
authors.lastname: Li
authors.affiliation: Alberta Provincial Laboratory for Public Health, Edmonton, Alberta, Canada School of Public Health, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Norman F
authors.initials: NF
authors.lastname: Neumann
authors.affiliation: Water Quality Services, The City of Calgary, Calgary, Alberta, Canada.
authors.forename: Norma
authors.initials: N
authors.lastname: Ruecker
authors.affiliation: New York City Department of Environmental Protection, Valhalla, New York, USA.
authors.forename: Kerri A
authors.initials: KA
authors.lastname: Alderisio
authors.affiliation: CH Diagnostic & Consulting Service, Berthoud, Colorado, USA.
authors.forename: Gregory D
authors.initials: GD
authors.lastname: Sturbaum
authors.affiliation: National Exposure Research Laboratory, U.S. Environmental Protection Agency, Cincinnati, Ohio, USA.
authors.forename: Eric N
authors.initials: EN
authors.lastname: Villegas
authors.affiliation: Cryptosporidium Reference Unit, Public Health Wales Microbiology, Singleton Hospital, Swansea, United Kingdom.
authors.forename: Rachel
authors.initials: R
authors.lastname: Chalmers
authors.affiliation: Australian Water Quality Centre, South Australian Water Corporation, Adelaide, South Australia, Australia.
authors.forename: Paul
authors.initials: P
authors.lastname: Monis
authors.affiliation: State Key Laboratory of Bioreactor Engineering, School of Resources and Environmental Engineering, East China University of Science and Technology, Shanghai, China.
authors.forename: Yaoyu
authors.initials: Y
authors.lastname: Feng
authors.affiliation: Division of Foodborne, Waterborne and Environmental Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA lxiao@cdc.gov.
authors.forename: Lihua
authors.initials: L
authors.lastname: Xiao
mesh_terms: D003457:Cryptosporidiosis / Q000469:parasitology*; D003458:Cryptosporidium / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D016054:DNA, Protozoan / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D005618:Fresh Water / Q000469:parasitology*; D005838:Genotype; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics; D006801:Humans; D058889:Molecular Typing / Q000379:methods*; D015800:Protozoan Proteins / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D018841:HSP90 Heat-Shock Proteins; D015800:Protozoan Proteins; D012337:RNA, Ribosomal, 18S