To improve the specificity ofdetecting Cryptosporidium in water, quantitative real-time PCR (qPCR) assays weredeveloped to detect and quantitate Cryptosporidium spp. oocysts in environmentalsamples; however, these approaches have varying degrees of specificity and sensitivityand were not specific to C. hominis or C. parvum. Therefore, the goal of thisproject was to improve upon current qPCR assays to specifically detect C. parvum, C.hominis, or all Cryptosporidium spp.The first goal of the experimental approach of this project was the development of three qPCR assays. The firstis a "pan-Crypto" qPCR assay that detects all Cryptosporidium species, while the secondand third assays specifically detect C. parvum or C. hominis oocysts, respectively.Preliminary results showed that the pan-Crypto qPCR assay can easily detect genomicDNA extracts from human infectious and animal forms of Cryptosporidium spp. like C.canis, C. felis, C. hominis, C. meleagridis, C. muris and C. parvum. Additionalexperiments further revealed that the pan-Crypto qPCR can detect levels as low as oneoocyst in reagent grade water. For the other qPCR assays developed, the C. parvumspecific qPCR only detected C. parvum oocysts and not C. canis, C. felis, C. hominis, C.meleagridis, or C. muris. Similarly, the C. hominis specific qPCR assay only detected C.hominis oocysts and not C. canis, C. felis, C. meleagridis, C. muris, or, C. parvum.Additional experiments that further evaluate limits of detection and specificities of allthree assays in detecting oocysts spiked in environmental samples are discussed. The second goal of the experimental approach of this project was to determine the variations of qPCR resultsgenerated from using different real-time PCR machines. To date, three real-time PCRmachines, ABI 7000, ABI 7900, and Roche LC 480 Smart Cycler, were compared.Preliminary results indicated that all three machines tested were able to detect low levelsof template DNA, equivalent to one oocyst, and no significant differences were observed.Performance results from other real-time PCR machines will also be presented as theybecome available. Includes 11 references, abstract only.
