Detection of a single viable Cryptosporidium parvum oocyst in environmental water concentrates by reverse transcription-PCR

Abstract

Current methods for detection of Cryptosporidium parvum oocysts in water are time-consuming and difficult. We have developed a reverse transcription (RT)-PCR which can detect the presence of a single viable oocyst spiked into concentrated environmental water samples. The test is based on the detection of mRNA from a C. parvum heat shock protein (hsp). The synthesis of hsp was induced by a short 45°C incubation followed by oocyst lysis by a freeze- thaw process. Hsp70 mRNA, produced only from viable oocysts, was then isolated by hybridization to oligo(dT)25-coated magnetic beads. Detection was achieved by RT-PCR amplification of a 590-bp region of hsp70 mRNA specific for C. parvum. To test th..