Giardia lamblia is one of the most recognized waterborne protozoan parasites causing
gastrointestinal disease. A simple but effective
DNA extraction protocol for real-time PCR detection from surface water samples was developed in this study. Eleven protocols were compared, which consisted of freeze-thaw treatments (liquid N(2) and boiling water) and purification using the Qiagen DNeasy kit, together with different combinations of
proteinase K, PVP360,
GITC and
Chelex 100 incubation. Using concentrated surface water samples spiked with G. lamblia
cysts, the necessary steps for high
DNA recovery were shown to be freeze-thaw, DNeasy purification and
Chelex 100 incubation. Multiple rounds of freeze-thaw treatment (five cycles per round) were reported for the first time in this study to significantly increase the
DNA yield from G. lamblia
cysts, from ~20% after one round of freeze-thaw to 40 and 70% after two and three-rounds of freeze-thaw, respectively. More than three rounds of freeze-thaw treatment did not promote additional
DNA recovery. The final protocol included three-three-rounds of freeze-thaw treatment, DNeasy purification and
Chelex 100 incubation. This method was simpler, more cost-effective, and had a comparable
DNA recovery to methods involving immunomagnetic separation.