Abstract
Real-time reverse transcription-polymerase chain reaction (RT-PCR) was applied to the detection of Cryptosporidium oocysts. Firstly, the sensitivity and specificity of three primer pairs were compared, and the primer pair reported by Miller et al. (2006) was considered to be the most suitable one. Secondly, a reverse transcription reaction was added to the real-time PCR assay for increase in sensitivity. The real-time RT-PCR assay revealed that one oocyst contains the 27,400 copies of rRNA, and the quantification limit of the assay was as low as 7.5×10－4 oocysts/test tube, while that of normal real-time PCR assay was as low as 2.4×10－1 oocysts/test tube. Therefore, the Cryptosporidium oocysts can theoretically be detected in reproducible tests using the real-time RT-PCR assay even in water samples containing only one oocyst. Thirdly, the developed real-time RT-PCR assay was applied to detect Cryptosporidium oocysts in 14 real river water samples. The concentration was quantified by the real-time RT-PCR assay, and was correlated to the suspected value, but not to the confirmed value, determined by conventional microscopic observation.
