Three sample preparation protocols for polymerase chain reaction based detection of Cryptosporidium parvum in environmental samples

Cryptosporidium parvum is a protozoan parasite responsible for an increasin g number of outbreaks of gastrointestinal illness worldwide. In this report , we describe development of sample preparation protocols for polymerase ch ain reaction (PCR)-based detection of C. parvum in fecal material and envir onmental water samples. Two of these methods were found adequate for isolat ion of Cryptosporidium DNA from filtered water pellet suspensions. The firs t involved several filtration steps, immunomagnetic separation and freeze-t haw cycles. The second method involved filtration, addition of EnviroAmp(TM ) lysis reagent, freeze-thaw cycles and precipitation of the DNA with isopr opanol. Using nested PCR, we detected 100 oocysts/ml of filtered water pell et suspension, with either of the above sample preparation procedures. Nest ed PCR increased sensitivity of the assay by two to three orders of magnitu de as compared to the primary PCR. The detection limit for seeded fecal sam ples was 10-fold higher than for filtered environmental water pellet suspen sion. Nested PCR results showed 62.4 and 91.1% correlation with immunofluor escence assay (LFA) for fecal samples and filtered environmental water pell et suspensions, respectively. This correlation decreased to 47.2% and 44.4% , respectively, when only IFA positive samples were analyzed. However, in f ecal samples contaminated with a high number (>10(5)/g) of C. parvum oocyst s, this correlation was 100%. (C) 1999 Elsevier Science B.V. All rights res erved.