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)

Naohiro Kishida, Ryo Miyata, Atsushi Furuta, Shinji Izumiyama, Satoshi Tsuneda, Yuji Sekiguchi, Naohiro Noda, Michihiro Akiba

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    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.

    Original languageEnglish
    Pages (from-to)187-194
    Number of pages8
    JournalWater Research
    Volume46
    Issue number1
    DOIs
    Publication statusPublished - 2012 Jan 1

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    Keywords

    • Cryptosporidiosis
    • Fluorescence quenching
    • Health-related water microbiology
    • Real-time PCR

    ASJC Scopus subject areas

    • Water Science and Technology
    • Waste Management and Disposal
    • Pollution
    • Ecological Modelling

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