Development of a direct DNA extraction protocol for real-time PCR detection of Giardia lamblia from surface water
- 434 Downloads
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.
KeywordsGiardia lamblia DNA extraction Real-time PCR Freeze–thaw Surface water
This research was completed while the first author was a Postdoctoral Fellow with the Natural Sciences and Engineering Research Council of Canada (NSERC) Chair in Water Treatment at the University of Waterloo. It was supported by the Ontario (Canada) Ministry of the Environment, Best in Science Program. The Partners of the NSERC Chair may be found at www.civil.uwaterloo.ca/watertreatment/.
- Anceno AJ, Katayama H, Houpt ER, Chavalitshewinkoon-Petmitr P, Chuluun Band Shipin OV (2007) IMS-free DNA extraction for the PCR-based quantification of Cryptosporidium parvum and Giardia lamblia in surface and waste water. Int J Environ Health Res 17(4):297–310. doi: 10.1080/09603120701372573 CrossRefGoogle Scholar
- Chung E, Aldom JE, Chagla AH, Kostrzynska M, Lee H, Palmateer G, Trevors JT, Ungerd S, De Grandis S (1998) Detection of Cryptosporidium parvum oocysts in municipal water samples by the polymerase chain reaction. J Microbiol Methods 33:171–180. doi: 10.1016/S0167-7012(98)00050-5 CrossRefGoogle Scholar
- Hallier-Soulier S, Guillot E (2003) An immunomagnetic separation-reverse transcription polymerase chain reaction (IMS-RT-PCR) test for sensitive and rapid detection of viable waterborne Cryptosporidium parvum. Environ Microbiol 5(7):592–598. doi: 10.1046/j.1462-2920.2003.00442.x CrossRefGoogle Scholar
- Rose JB, Slifko TRG (1999) Cryptosporidium, and Cyclospora and their impact on foods: a review. J Food Prot 64:1793–1798Google Scholar
- Tanrıverdi S, Tanyeli A, Baslamish F, Koksal F, Kılınc Y, Feng X, Batzer G, Tzipori S, Widmer G (2002) Detection and genotyping of oocysts of Cryptosporidium parvum by real-time PCR and melting curve analysis. J Clin Microbiol 40(9):3237–3324. doi: 10.1128/JCM.40.9.3237-3244.2002 CrossRefGoogle Scholar
- US Environmental Protection Agency (2005) Method 1623: Cryptosporidium and Giardia in water by filtration, immunomagnetic separation, and fluorescent antibody. Office of Water, Washington, DC. Publication EPA-821-R-99-006Google Scholar
- Verweij JJ, Blangé RA, Templeton K, Schinkel J, Brienen EAT, van Rooyen MAA, van Lieshout L, Polderman AM (2004) Simultaneous detection of Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum in fecal samples by using multiplex real-time PCR. J Clin Microbiol 42(3):1220–1223. doi: 10.1128/JCM.42.3.1220-1223.2004 CrossRefGoogle Scholar