, Volume 18, Issue 6, pp 661–668 | Cite as

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

  • Xin Yu
  • Michele I. Van Dyke
  • Andrea Portt
  • Peter M. Huck


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.


Giardia 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


  1. Alberti S, Fornaro M (1990) Higher transaction efficiency of genomic DNA purified with a guanidinium thiocyanate-based procedure. Nucleic Acids Res 18(2):351–353. doi: 10.1093/nar/18.2.351 CrossRefGoogle Scholar
  2. Amar CFL, Dear PH, McLauchlin J (2003) Detection and genotyping by real-time PCR/RFLP analyses of Giardia duodenalis from human faeces. J Med Microbiol 52(8):681–683. doi: 10.1099/jmm.0.05193-0 CrossRefGoogle Scholar
  3. 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
  4. Betancourt WQ, Rose JB (2004) Drinking water treatment processes for removal of Cryptosporidium and Giardia. Vet Parasitol 126:219–234CrossRefGoogle Scholar
  5. 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
  6. Fontaine M, Guillot E (2002) Development of a taqMan quantitative PCR assay specific for Cryptosporidium parvum. FEMS Microbiol Lett 214:13–17. doi: 10.1111/j.1574-6968.2002.tb11318.x CrossRefGoogle Scholar
  7. Fontaine M, Guillot E (2003) An immunomagnetic separation-real-time PCR method for quantification of Cryptosporidium parvum in water samples. J Microbiol Methods 54:29–36. doi: 10.1016/S0167-7012(03)00005-8 CrossRefGoogle Scholar
  8. Ghosh S, Debnath A, Sil A, Chattopadhyay DJ, Das P (2000) PCR detection of Giardia lamblia in stool: targeting intergenic spacer region of multicopy rRNA gene. Mol Cell Probes 14(3):181–189. doi: 10.1006/mcpr.2000.0302 CrossRefGoogle Scholar
  9. Guy RA, Payment P, Krull UJ, Horgen PA (2003) Real-time PCR for quantification of Giardia and Cryptosporidium in environmental water samples and sewage. Appl Environ Microbiol 69:5178–5185. doi: 10.1128/AEM.69.9.5178-5185.2003 CrossRefGoogle Scholar
  10. Guy RA, Xiao C, Horgrn PA (2004) Real-time PCR assay for detection and genotype differentiation of Giardia lamblia in stool specimens. J Clin Microbiol 42(7):3317–3320. doi: 10.1128/JCM.42.7.3317-3320.2004 CrossRefGoogle Scholar
  11. 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
  12. Hu J, Feng Y, Ong SL, Ng WJ, Song L (2004) Improvement of recoveries for the determination of protozoa Cryptosporidium and Giardia in water using method 1623. J Microbiol Methods 58:321–325. doi: 10.1016/j.mimet.2004.04.013 CrossRefGoogle Scholar
  13. Kotlowski R, Martin A, Ablordey A, Chemlal K, Fonteyne P, Portaels F (2004) One-tube cell lysis and DNA extraction procedure for PCR-based detection of Mycobacterium ulcerans in aquatic insects, molluscs and fish. J Med Microbiol 53:927–933. doi: 10.1099/jmm.0.45593-0 CrossRefGoogle Scholar
  14. Lane S, Lloyd D (2002) Current trends in research into the waterborne parasite Giardia. Crit Rev Microbiol 28(2):123–147. doi: 10.1080/1040-840291046713 CrossRefGoogle Scholar
  15. Ramirez NE, Sreevatsan S (2006) Development of a sensitive detection system for Cryptosporidium in environmental samples. Vet Parasitol 136:201–213. doi: 10.1016/j.vetpar.2005.11.023 CrossRefGoogle Scholar
  16. Rose JB, Slifko TRG (1999) Cryptosporidium, and Cyclospora and their impact on foods: a review. J Food Prot 64:1793–1798Google Scholar
  17. 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
  18. 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
  19. Verweij JJ, Schinkel J, Laeijendecker D, van Rooyen MAA, van Lieshout L, Polderman AM (2003) Real-time PCR for the detection of Giardia lamblia. Mol Cell Probes 17:223–225. doi: 10.1016/S0890-8508(03)00057-4 CrossRefGoogle Scholar
  20. 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
  21. Wang Z, Vora GJ, Stenger DA (2004) Detection and genotyping of Entamoeba histolytica, Entamoeba dispar, Giardia lamblia, and Cryptosporidium parvum by oligonucleotide microarray. J Clin Microbiol 42(7):3262–3271. doi: 10.1128/JCM.42.7.3262-3271.2004 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Xin Yu
    • 1
  • Michele I. Van Dyke
    • 2
  • Andrea Portt
    • 2
  • Peter M. Huck
    • 2
  1. 1.Institute of Urban EnvironmentChinese Academy of SciencesXiamenChina
  2. 2.Department of Civil and Environmental EngineeringUniversity of WaterlooWaterlooCanada

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