AWWA WQTC55007

The public health problem posed by Cryptosporidium parvum has urged the water supply industry to develop very accurate analytical tools to assess the presence of this parasite in water. The classical method based on indirect immunofluorescence assay (IFA) routinely used for the detection of Cryptosporidium oocysts in water is time-consuming and non-specific for C. parvum, the species which affects humans. In order to circumvent these limitations, a quantitative test based on real-time quantitative PCR using the TaqMan fluorogenic detection system (TaqMan PCR) was developed. TaqMan PCR identified a 138 base pair section of a Cryptosporidium parvum genomic sequence by using a specific fluorogenic probe and two primers. This system used the high performance of DNA polymerase for nucleic amplification and a 5'nuclease assay with an internal fluorogenic DNA probe for real-time quantitation. Fluorescence intensity was directly proportional to the copy number of the C. parvum target DNA sequence and was detected with an automated fluorometer. Quantitation was accomplished by comparing the fluorescence signals obtained from samples with unknown numbers of C. parvum oocysts with fluorescence signals from C. parvum oocysts standard dilutions. This real time quantitative PCR assay allows reliable quantitation of C. parvum oocysts over a 6-log dynamic range within three hours. This molecular test will be very useful for the water industry; it can be used for drinking water quality control as well as for the evaluation of treatment efficiency and the identification of risk resources. Includes 11 references, figures.