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

Since 1991 more than 30 PCR protocols have been published, which show a pot ential to replace the current microscopic detection method for Cryptosporid ium parvum in environmental samples and food. This review provides a synopt ic comparison of these protocols with respect to the following features: is olation and purification of oocysts from tested matrices, elimination of fr ee DNA, viability and infectivity assessment, release of nucleic acids, nuc leic acid extraction, type of PCR (PCR, RT-PCR, internal-standard-PCR, in s itu PCR, TaqMan-PCR), primary product detection, additional specificity con trol, secondary product detection, reported sensitivity, cross-reaction wit h other Cryptosporidium species, and target and sequence information such a s amplicon length, primer sequences, multiple copy target, presence of stra in-specific differences in the amplicon, GenBank accession numbers and gene function. The results demonstrate that problems like PCR inhibition, viabi lity assessment, and the requirement of an extreme sensitivity have been so lved. PCR assays would be most valuable to control presence-absence standar ds in defined matrix volumes, and the setup of such standards would very mu ch contribute to a rapid introduction of this awaited technology into routi ne monitoring of environmental, water and food samples, and to a further st andardization of the Various protocols. It can be expected that satisfactor y solutions for quantification will be found for a growing number of PCR-ba sed assays. Systematic field evaluation and interlaboratory studies will co mplement our present knowledge of these methods in the near future.