PCR-based quantitation of Cryptosporidium parvum in municipal water samples

A PCR method for the quantitation of Cryptosporidium parvum oocysts in muni cipal drinking water samples was investigated. Quantitative PCR uses an int ernal standard (IS) template with unknown target numbers to compare to stan dards of known concentrations in a standard curve. The IS template was ampl ified using the same primers used to amplify a portion of a 358 bp gene fra gment that encodes a repetitive oocyst wall protein in C. parvum. Municipal water samples spiked with known numbers of C. parvum oocysts were tested b y quantitative PCR using the IS and the Digene SHARP Signal(TM) System Assa y for PCR product detection. The absorbance readings for target DNA and IS templates versus the number of molecules of the target DNA were plotted to generate standard curves for estimating oocyst numbers. The method allowed the quantitation of oocysts from log 3 to log 5 spiked into municipal water samples. (C) 1999 Elsevier Science B.V. All rights reserved.