Methods

First we describe several methods for extracting DNA from Cryptosporidium oocysts for PCR analysis. These methods are suitable for extracting DNA directly from fecal samples or from environmental samples containing oocysts. Two sample preparation methods are discussed: (1) direct extraction of fecal/environmental DNA; and (2) isolation of oocysts followed by DNA extraction. Direct extraction of DNA is faster, but we frequently prefer to purify the oocysts first, as a portion of these oocysts can subsequently be used for additional studies or animal propagation. Extracting DNA from purified oocysts also reduces the probability of contamination of the DNA with PCR inhibitors. Second, two PCR-based methods for analyzing microsatellite polymorphisms in Cryptosporidium are discussed: (1) traditional PCR, and (2) real-time PCR.

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