Molecular-based methods for the detection and quantification of infectious Cryptosporidium parvum oocysts in environmental samples.

摘要

The protozoan parasite Cryptosporidium parvum occurs widely in both raw and treated drinking water, and is responsible for numerous cryptosporidiosis outbreaks worldwide. Because detecting the presence of C. parvum does not assess oocyst viability or infectivity, current quantitative detection strategies have been coupled with in vitro culture methods to assess oocyst infectivity and viability. Immunological and molecular-based methods were evaluated for their ability to quantify infection of immunomagnetic separation (IMS)-recovered oocysts from seeded raw water concentrates. Most probable number cell culture polymerase chain reaction (MPN-CC-PCR) and most probable number-foci detection method (MPN-FDM) estimates for each seed dose were generally within a one-log unit of directly enumerated foci of infection. Real-time TaqMan and competitive PCR results were within a half-log and greater than one-log range of directly enumerated foci, respectively. Real-time PCR provided the highest sensitivity, with the ability to detect as few as 41 infectious oocysts from a log 2 seed spike into 10 L raw water concentrate.