Comparison of propidium monoazide-quantitative PCR and reverse transcription quantitative PCR for viability detection of fresh Cryptosporidium oocysts following disinfection and after long-term storage in water samples

Abstract

Purified oocysts of Cryptosporidium parvum were used to evaluate the applicability of two quantitative PCR (qPCR) viability detection methods in raw surface water and disinfection treated water. Propidium monoazide-qPCR targeting hsp70 gene was compared to reverse transcription (RT)-qPCR heat induced hsp70 mRNA in water samples spiked with oocysts. Changes in viability of flow cytometry sorted fresh and oocysts having undergone various aging periods (up to 48 months at 4 °C) were evaluated by Ct values obtained from the qPCR before and after disinfection scenarios involving ammonia or hydrogen peroxide. Both qPCR methods achieved stability in dose dependent responses by hydrogen peroxide treatment in distilled water that proved their suitability for the viability evaluations. Oocysts exposed to 3% hydrogen peroxide were inactivated at a rate of 0.26 h−1 and 0.93 h−1, as measured by the mRNA assay and the PMA-DNA assay, respectively. In contrast, the PMA-DNA assay was not as sensitive as the mRNA assay in detecting viability alterations followed by exposure to ammonia or after a long-term storage in 4 °C in distilled water since no dose response dependency was achieved. Surface water concentrates containing enhanced suspendable solids determined that changes in viability were frequently detected only by the mRNA method. Failure of, or inconsistency in the detection of oocysts viability with the PMA-DNA method, apparently resulted from solids that might have reduced light penetration through the samples, and thus inhibited the cross-linking step of PMA-DNA assay.