AWWA WQTC69416

The goal of this project was to develop a rapid molecular technique that can specifically detect and genotype viable Cryptosporidium oocysts. The authors have developed and optimized the use of propidium monoazide (PMA) with a PCR-based assay that enables detection and genotyping of viable oocysts. PMA is a DNA intercalating dye that only penetrates dead or damaged cells. Unlike other intercalating "vital" dyes, PMA can covalently bind to DNA upon exposure to bright light. As a result, only the genomic DNA from viable cells, which is not intercalated with PMA, is retained following DNA extraction procedures and can subsequently be detected by PCR analysis. To date, PMA has only been used to differentiate live/dead bacteria and fungi and has not yet been applied to protozoan parasites. Results in this paper show that heat killed oocysts treated with PMA were not detected while live oocysts treated with PMA were detected using conventional PCR. Includes 7 references.