PCR detection of Cryptosporidium parvum in environmental samples--a review of published protocols and current developments

Abstract

Since 1991 more than 30 PCR protocols have been published, which show a potential to replace the current microscopic detection method for Cryptosporidium parvum in environmental samples and food. This review provides a synoptic comparison of these protocols with respect to the following features: isolation and purification of oocysts from tested matrices, elimination of free DNA, viability and infectivity assessment, release of nucleic acids, nucleic acid extraction, type of PCR (PCR, RT-PCR, internal-standard-PCR, in situ PCR, TaqMan-PCR), primary product detection, additional specificity control, secondary product detection, reported sensitivity, cross-reaction with other Cryptosporidium species, and target and sequence information such as amplicon length, primer sequences, multiple copy target, presence of strain-specific differences in the amplicon, GenBank accession numbers and gene function. The results demonstrate that problems like PCR inhibition, viability assessment, and the requirement of an extreme sensitivity have been solved. PCR assays would be most valuable to control presence-absence standards in defined matrix volumes, and the setup of such standards would very much contribute to a rapid introduction of this awaited technology into routine monitoring of environmental, water and food samples, and to a further standardization of the various protocols. It can be expected that satisfactory solutions for quantification will be found for a growing number of PCR-based assays. Systematic field evaluation and interlaboratory studies will complement our present knowledge of these methods in the near future.