Recovery of Cryptosporidium from spiked water and stool samples measured by PCR and real time PCR
2012
Adamska, M., University of Szczecin (Poland). Dept. of Genetics | Leonska-Duniec, A., University of Szczecin (Poland). Dept. of Genetics | Sawczuk, M., University of Szczecin (Poland). Dept. of Genetics | Maciejewska, A., University of Szczecin (Poland). Dept. of Genetics | Skotarczak, B., University of Szczecin (Poland). Dept. of Genetics
We carried out trials in order to assess the effectiveness of recovery of Cryptosporidium parvum oocysts from spiked environmental and distilled water samples, filtrated and concentrated with the use of special laboratory equipment. Inactivation of inhibitors was carried out with use of bovine serum albumin (BSA) in PCR mixes at ten different concentrations. DNA extraction was carried out from stool samples spiked with C. parvum oocysts, concentrated using two methods, and unconcentrated. Nested PCR and a TaqMan nested real time PCR assay, targeting the 18S rRNA gene, was used to detect C. parvum DNA in spiked water and additionally in spiked stool samples. The obtained results showed that losses of C. parvum oocysts occur during the filtration and concentration of spiked water samples. The addition of small amounts of BSA (5-20 ng/microL) to PCR and TaqMan PCR mixes increases the sensitivity of both methods, but a high concentration of BSA (100 ng/microL and above) has an inhibiting effect on the polymerase reaction. The extraction of DNA from C. parvum oocysts from spiked stool samples preceded by concentration with PBS, ether and Percoll resulted in a higher copy number of the 18S rRNA gene.
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