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Detection of Cryptosporidium parvum in Secondary Effluents Using a Most Probable Number–Polymerase Chain Reaction Assay
Author(s) -
Tsuchihashi Ryujiro,
Loge Frank J.,
Darby Jeannie L.
Publication year - 2003
Publication title -
water environment research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.356
H-Index - 73
eISSN - 1554-7531
pISSN - 1061-4303
DOI - 10.2175/106143003x141097
Subject(s) - cryptosporidium parvum , serial dilution , cryptosporidium , effluent , microbiology and biotechnology , polymerase chain reaction , biology , chromatography , detection limit , wastewater , chemistry , environmental engineering , feces , environmental science , biochemistry , medicine , alternative medicine , pathology , gene
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.