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

Abstract

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 hsp70 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.

Cryptosporidium Propidium Monoazide-PCR, a Molecular Biology-Based Technique for Genotyping of Viable Cryptosporidium Oocysts

“…Other more recent approaches utilize molecular biology-based techniques to assess oocyst viability. Fluorescence in situ hybridization (5,10,20,26,40,47), nucleic acid sequence-based amplification (4), reverse transcriptase PCR (RT-PCR) (17,22,50), and an integrated cell culture/ PCR assay (12,25) all show promise in providing a rapid and relatively inexpensive viability assay for Cryptosporidium. However, it still remains to be determined if these methods can detect all Cryptosporidium species and genotypes found in the environment.…”

Oligonucleotide-Gold Nanoparticle Networks for Detection of Cryptosporidium parvum Heat Shock Protein 70 mRNA

“…Previous studies indicate a 1,000-to 10,000-fold increase in the amount of HSP70 mRNA recovered from Drosophila species under heat stress (14). Second, HSP70 mRNA is a good indicator of oocyst viability; only viable oocysts will produced HSP70 mRNA by the use of heat shock (9,19). Third, previous studies suggest that the half-life of HSP70 mRNA (ϳ50 min) extracted from human cell lines (HeLa and 283 cells) is increased at least 10-fold upon heat shock, suggesting the increased stability of the targets (21).…”

Overview of Cryptosporidium Presentations at the 10th International Workshops on Opportunistic Protists

“…Therefore, more recent efforts have been concentrated on reverse transcriptase PCR (RT-PCR) detection of mRNA, which degenerates more quickly than rRNA (35). Several targeted genes have been used, including the ␤-tubulin, hsp70, amyloglucosidase, and CP2 genes (18,22,27,35). A current challenge is the improvement of sensitivity of RT-PCR detection protocols.…”

“…A current challenge is the improvement of sensitivity of RT-PCR detection protocols. Although the detection limit of one viable oocyst was achieved in initial studies, much higher limits (10 to 1,000 viable oocysts per sample) were reported subsequently (22,27,35), and in at least one instance, the decay of mRNA to below limits of detection preceded the loss of oocyst infectivity in cell culture and mouse models (26). Others have used nucleic acid sequence-based amplification of Cryptosporidium mRNA in assessments of the viability of oocysts in water samples (12).…”