A PCR method for the quantitation of Cryptosporidium parvum oocysts in municipal drinking water samples was investigated. Quantitative PCR uses an internal standard (IS) template with unknown target numbers to compare to standards of known concentrations in a standard curve. The IS template was amplified using the same primers used to amplify a portion of a 358 bp gene fragment that encodes a repetitive oocyst wall protein in C. parvum. Municipal water samples spiked with known numbers of C. parvum oocysts were tested by quantitative PCR using the IS and the Digene SHARP Signal System Assay 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.

The enteric protozoan, Cryptosporidium parvum, the causative agent for cryptosporidiosis, has been isolated from drinking water, fecal samples from humans and animals, and environmental samples such as sediment and soil. The currently available water sampling methods for detection of this parasite are labor-intensive and the efficiency of oocyst recovery is poor. A recent improved method utilizing membrane filtration and dissolution followed by polymerase chain reaction (PCR) amplification, and confirmatory nested PCR was evaluated for the sensitive and specific detection of C. parvum oocysts. Detection of PCR products by the ELISA-based Digene SHARP Signal™ System Assay was assessed for sensitivity. Seventy-two municipal water samples ranging in volume from 230 to 1,000 l from southwestern Ontario, Canada were spiked with varying concentrations of formalin-killed C. parvum oocysts for use in this study. Oocyst recovery on the filters was determined by the Merifluor immunofluoresence assay for Cryptosporidium/Giardia. Oocyst detection using the PCR assay showed an 84.7% correlation with immunofluoresence assay (IFA) results. During optimization studies, the correlation between PCR and IFA reached 98%. The sensitivity of a primary PCR assay ranged from 1 to 10 oocysts per reaction, which was equivalent to 102 to 103 oocysts per 100 l municipal water. The PCR assay also showed potential for application to untreated water samples and naturally contaminated municipal water from a recent Cryptosporidium outbreak. Further application of nested PCR may improve overall sensitivity and specificity for detecting C. parvum in municipal water samples since combined primary and nested PCR results showed 97.2% correlation with IFA. The Digene SHARP Signal™ System assay offers a sensitive and specific alternative for detection of C. parvum amplification products.

In this opinion paper, we offer our perspective on our bioremediation research along with the methods to assess its effectiveness as a safe and beneficial technology to remediate selected soil sites. The isolation and characterization of bacterial isolates from chemically contaminated soils, their survival and catabolic activity in contaminated soil, toxicity testing in chemically contaminated soils, molecular-based methods of detection such as the polymerase chain reaction (PCR) and DNA probing are discussed. By using numerous conventional microbiological, chemical techniques and molecular based methods, bioremediation can be studied in a comprehensive manner and the technology transferred to the commercial sector.