title: Development of a direct DNA extraction protocol for real-time PCR detection of Giardia lamblia from surface water.
abstract: 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.
authors.affiliation: Institute of Urban Environment, Chinese Academy of Sciences, 1799 Jimei Avenue, 361021 Xiamen, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Yu
authors.forename: Michele I
authors.initials: MI
authors.lastname: Van Dyke
authors.forename: Andrea
authors.initials: A
authors.lastname: Portt
authors.forename: Peter M
authors.initials: PM
authors.lastname: Huck
mesh_terms: D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification*; D004784:Environmental Monitoring / Q000379:methods*; D005615:Freezing; D005618:Fresh Water / Q000469:parasitology*; D016829:Giardia lamblia / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D016054:DNA, Protozoan