NDLI: Development of a direct DNA extraction protocol for real-time PCR detection of Giardia lamblia from surface water

Content Provider	Springer Nature Link
Author	Yu, Xin Dyke, Michele I. Portt, Andrea Huck, Peter M.
Copyright Year	2009
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 N2 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.
Starting Page	661
Ending Page	668
Page Count	8
File Format	PDF
ISSN	09639292
Journal	Ecotoxicology
Volume Number	18
Issue Number	6
e-ISSN	15733017
Language	English
Publisher	Springer US
Publisher Date	2009-06-05
Publisher Place	Boston
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Giardia lamblia DNA extraction Real-time PCR Freeze–thaw Surface water Environmental Management Ecology Environment Ecotoxicology
Content Type	Text
Resource Type	Article
Subject	Health, Toxicology and Mutagenesis Medicine Toxicology Management, Monitoring, Policy and Law

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