Winning Abstracts from the 2006 National Medical Student Abstract Competition: Detection and Quantification of Cryptosporidium Parvum in HCT-8 Cells and Human Fecal Specimens using Real-time Polymerase Chain Reaction Assay.
Author: Jonathan B. Parr, University of Virginia School of Medicine, 2008
Cryptosporidium parvum is a significant cause of diarrheal illness worldwide, especially among children and immunocompromised patients. Currently used diagnostic techniques are time-consuming, require skilled technicians, and are not useful for quantification of oocysts in fecal and environmental samples.
In this study, we examined the utility of a real-time polymerase chain reaction (PCR) assay for detecting and quantifying Cryptosporidium parvum oocysts in three distinct sets of samples: phosphate buffered saline (PBS), HCT-8 cells (human ileocecal carcinoma), and human fecal specimens. A reliable standard curve was generated using the PBS samples spiked with pure oocysts, and oocyst starting quantities were calculated for the infected HCT-8 cell and spiked fecal samples.
The average difference between known and calculated starting quantities was 1.4±0.3 logs oocysts/sample for the HCT-8 cells and 2.2±0.5 logs oocysts/sample for the spiked stool samples. Despite these losses, the assay detected oocysts in HCT-8 cells and fecal specimens infected/spiked with at least 3 logs oocysts/sample.
Our results confirm that real-time PCR can be used to detect and quantify Cryptosporidium parvum oocysts in a variety of samples in the research laboratory and that it will likely prove to be a useful tool in the field.
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