Detection of Cryptosporidium parvum and C. muris Oocysts in Spiked Backwash Water using Three PCR-Based Protocols

Abstract

Cryptosporidium parvum is an important protozoan that was shown in recent years to be responsible for a number of water-borne outbreaks of diarrhoea. In this study, ways to improve the extraction of DNA from purified C. parvum oocysts and from backwash water (a heavily contaminated by-product of sand filtration), for use in the polymerase chain reaction (PCR), were investigated. The use of a commercial DNA purification kit reduced overall assay time as did the inclusion of an excystation step. In addition, a comparison was made between the detection limits of three PCR based protocols (standard, nested and arbitrary primed (AP)-PCR) for the detection of C. parvum and C. muris. The three PCR protocols were assayed using serially diluted DNA extracted from purified C. parvum and C. muris oocysts, and from backwash water spiked with known numbers of C. parvum oocysts. Nested PCR was the most sensitive PCR-based method tested for detecting C. parvum DNA followed by AP-PCR and standard PCR. Therefore, a system based on nested PCR for the routine monitoring of backwash water may act as a first-line detection test for the presence of C. parvum oocysts in raw water treated for human consumption.