title: Quantitative detection of Cryptosporidium oocyst in water source based on 18S rRNA by alternately binding probe competitive reverse transcription polymerase chain reaction (ABC-RT-PCR).
abstract: We describe an assay for simple and cost-effective quantification of Cryptosporidium oocysts in water samples using a recently developed quantification method named alternately binding probe competitive PCR (ABC-PCR). The assay is based on the detection of 18S rRNA specific for Cryptosporidium oocysts. The standard curve of the ABC-PCR assay had a good fitting to a rectangular hyperbola with a correlation coefficient (R) of 0.9997. Concentrations of Cryptosporidium oocysts in real river water samples were successfully quantified by the ABC-reverse transcription (RT)-PCR assay. The quantified values by the ABC-RT-PCR assay very closely resemble those by the real-time RT-PCR assay. In addition, the quantified concentration in most water samples by the ABC-RT-PCR assay was comparable to that by conventional microscopic observation. Thus, Cryptosporidium oocysts in water samples can be accurately and specifically determined by the ABC-RT-PCR assay. As the only equipment that is needed for this end-point fluorescence assay is a simple fluorometer and a relatively inexpensive thermal cycler, this method can markedly reduce time and cost to quantify Cryptosporidium oocysts and other health-related water microorganisms.
authors.affiliation: Division of Water Management, Department of Environmental Health, National Institute of Public Health, 2-3-6 Minami, Wako, Saitama 351 0197, Japan. kishida@niph.go.jp
authors.forename: Naohiro
authors.initials: N
authors.lastname: Kishida
authors.forename: Ryo
authors.initials: R
authors.lastname: Miyata
authors.forename: Atsushi
authors.initials: A
authors.lastname: Furuta
authors.forename: Shinji
authors.initials: S
authors.lastname: Izumiyama
authors.forename: Satoshi
authors.initials: S
authors.lastname: Tsuneda
authors.forename: Yuji
authors.initials: Y
authors.lastname: Sekiguchi
authors.forename: Naohiro
authors.initials: N
authors.lastname: Noda
authors.forename: Michihiro
authors.initials: M
authors.lastname: Akiba
mesh_terms: D003458:Cryptosporidium / Q000235:genetics* / Q000302:isolation & purification*; D007564:Japan; D033661:Oocysts / Q000378:metabolism*; D015347:RNA Probes / Q000378:metabolism*; D012337:RNA, Ribosomal, 18S / Q000032:analysis* / Q000235:genetics; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014867:Water / Q000469:parasitology*
chemical_list: D015347:RNA Probes; D012337:RNA, Ribosomal, 18S; D014867:Water