Comparison of simple DNA extraction procedures to detect protozoa in a water sample by real-time PCR

Giardia and Cryptosporidium are parasitic protozoa responsible for enteritis in man, often severe in children and in immunocompromised patients. Their transmissive stages, cysts and oocysts, voided in large number with the faeces of infected hosts, may contaminate sewage effluents and surface water. Detection of these microorganisms in water environmental samples is a critical issue. Conventional methods for detection of Cryptosporidium parvum and Giardia lamblia are labor-intensive and time-consuming. Molecular detection methods based on amplification techniques have been applied for water quality management, but require a previous protozoa enrichment step by filtration and elution. Direct environmental DNA extraction plays a key role, but is limited by copurification of polymerase inhibitors. We have compared lysis and DNA isolation methods for protozoa detection by real-time per. The molecular approach provides a promising and effective tool for flanking the traditional optical microscopy based assays.