Enrichment of Cryptosporidium parvum from in vitro culture as measured by total RNA and subsequent sequence analysis

摘要

Cryptosporidium parvum is an apicomplexan parasite that infects a wide range of hosts including humans. Due to the parasite's quasi-intracellular, intermembrane location on the host cell, it is difficult to purify parasites from in vitro and in vivo infections for molecular studies. We have developed a method to greatly enrich in vitro C. parvum merozoites from host cells. The efficiency of the protocol was assessed with C. parvum (KSU-1 isolate) parasites of different developmental stages isolated following a synchronized infection of HCT-8 host cells. Total RNA was extracted from the samples and used to evaluate the quantity of host cell contamination in enriched parasite fractions. The quality of the RNA was verified using an Agilent BioAnalyzer. cDNA libraries of RNA isolated from 24 and 48 h C. parvum in vitro preparations isolated via this protocol were sequenced at the Broad Institute via an NIH Microbial Sequencing (GSCID) Contract. Cryptosporidium sequences comprised 30% of the cDNA reads, demonstrating significant enrichment.