An immunomagnetic separation-reverse transcription polymerase chain reaction (IMS-RT-PCR) test for sensitive and rapid detection of viable waterborne Cryptosporidium parvum

The public health problem posed by the waterborne parasite Cryptosporidium parvum incited the water supply industry to develop very accurate analytical tools able to assess the presence of viable oocysts in drinking water. In this study, we report the development of a viability assay for C. parvum oocysts based on immunomagnetic separation and reverse transcription polymerase chain reaction (IMS-RT-PCR). The detection limit of the IMS-RT-PCR assay, which targets the hsp 70 heat shock-induced mRNA, was in the range of ten viable oocysts per 100-l tap water samples. Purified Cryptosporidium parvum oocysts were exposed to heating, freezing and three chemical disinfection treatments namely, chlorination, chlorine dioxide treatment and ozonation under conventional doses used in water treatment plants, then detected by IMS-PCR and IMS-RT-PCR. The results obtained by IMS-PCR showed that none of the treatments had an effect on oocyst detection. The inactivation of oocysts by boiling resulted in no RT-PCR signal. Chlorine as well as chlorine dioxide did not influence oocyst viability as determined by IMS-RT-PCR. Ozone more effectively inactivated oocysts. The IMS-RT-PCR assay in conjunction with IMS-PCR marks the development of a combined detection and viability test which can be used for drinking water quality control as well as for reliable evaluation of treatment efficiency.