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

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Edition: Vol. - No. Published: 11/01/2008 Number of Pages: 3File Size: 1 file , 700 KB