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In an attempt to improve the methodology for assessing the viability of C. parvum oocysts, the presence of specific RNA transcripts in oocysts was studied. The rationale for this approach is based on the observation that cellular RNA is subject to rapid turnover. In dead cells, in the absence of de novo synthesis, RNA levels decline rapidly as a consequence of nucleolytic degradation. The presence in oocysts of two messenger RNA transcripts and of the small subunit ribosomal RNA (SSU rRNA) was investigated in heat inactivated and naturally aged oocysts. Reverse-transcription followed by PCR amplification was initially attempted using primers specific for the SSU rRNA transcript. Using a single 35-cycle amplification protocol SSU rRNA was consistently detected in live as well as in inactivated oocysts. The persistence in our assay of rRNA in non-viable oocysts made this target unsuitable for the purpose of a viability assay. We assume that rRNA persists post-mortem because it is incorporated in ribosomes and therefore, less exposed to nucleases. Includes 10 references.

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Edition: Vol. - No. Published: 01/01/1999File Size: 1 file , 220 KB