Detection of Cryptosporidium parvum in Secondary Effluents Using a Most Probable Number–Polymerase Chain Reaction Assay
Authors: Tsuchihashi, Ryujiro; Loge, Frank J.; Darby, Jeannie L.
Source: Water Environment Research, Volume 75, Number 4, July/August 2003 , pp. 292-299(8)
Publisher: Water Environment Federation
Abstract:Polymerase chain reaction (PCR) was used to detect Cryptosporidium parvum oocysts in secondary effluent samples collected from activated-sludge facilities. Serial dilutions of the purified nucleic acid extracts from the samples were made and PCR was conducted to estimate the C. parvum oocyst concentration via a Poisson distribution-based most probable number (MPN). The degree of oocysts associated with wastewater particles was also evaluated. The sensitivity of the MPN–PCR assay was 20 oocysts/PCR unit. The detection limit of the concentration, extraction, and purification protocols in phosphate buffer saline spiked with a known concentration of oocysts ranged from 1.1 to 4.6 oocysts/L; the detection limit for the wastewater samples ranged from 11 to 4200 oocysts/L depending on the extent of inhibition in each sample. The recovery efficiency of the oocysts ranged from 48 to 59% in most samples. Oocysts were found in two out of seven samples with concentrations of 203 and 308 oocysts/L, as estimated by the MPN–PCR method. The oocysts were found only in the filtrate of the grab samples; particle-associated oocysts were not detected. Association of spiked C. parvum oocysts with particles in secondary effluent drawn from wastewater plants with varying operating conditions indicated a weak correlation between the degree of association and the mean cell residence time of the system.
Document Type: Research Article
Publication date: July 1, 2003
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