An easy 'one tube' method to estimate viability of Cryptosporidium oocysts using real-time qPCR.

Viability estimation of the highly resistant oocysts of Cryptosporidium remains a key issue for the monitoring and control of this pathogen. We present here a simple 'one tube' quantitative PCR (qPCR) protocol for viability estimation using a DNA extraction protocol which preferentially solubilizes excysted sporozoites rather than oocysts. Parasite DNA released from excysted sporozoites was quantified by real-time qPCR using a ribosomal DNA marker. The qPCR signal was directly proportional to the number of oocysts excysted, and a power-law relationship was noted between oocyst age and the proportion excysting. Unexcysted oocysts released negligible amounts of DNA making the method suitable for estimating viability of as few as 10 oocysts.