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Applied and Environmental Microbiology, September 2003, p. 5178-5185, Vol. 69, No. 9
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.9.5178-5185.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Real-Time PCR for Quantification of Giardia and Cryptosporidium in Environmental Water Samples and Sewage

Rebecca A. Guy,1* Pierre Payment,2 Ulrich J. Krull,1 and Paul A. Horgen1

University of Toronto at Mississauga, Mississauga, Ontario, Canada L5L 1C6,1 INRS-Institut Armand-Frappier, Laval, Quebec, Canada H7V 1B72

Received 5 March 2003/ Accepted 23 June 2003

The protozoan pathogens Giardia lamblia and Cryptosporidium parvum are major causes of waterborne enteric disease throughout the world. Improved detection methods that are very sensitive and rapid are urgently needed. This is especially the case for analysis of environmental water samples in which the densities of Giardia and Cryptosporidium are very low. Primers and TaqMan probes based on the ß-giardin gene of G. lamblia and the COWP gene of C. parvum were developed and used to detect DNA concentrations over a range of 7 orders of magnitude. It was possible to detect DNA to the equivalent of a single cyst of G. lamblia and one oocyst of C. parvum. A multiplex real-time PCR (qPCR) assay for simultaneous detection of G. lamblia and C. parvum resulted in comparable levels of detection. Comparison of DNA extraction methodologies to maximize DNA yield from cysts and oocysts determined that a combination of freeze-thaw, sonication, and purification using the DNeasy kit (Qiagen) provided a highly efficient method. Sampling of four environmental water bodies revealed variation in qPCR inhibitors in 2-liter concentrates. A methodology for dealing with qPCR inhibitors that involved the use of Chelex 100 and PVP 360 was developed. It was possible to detect and quantify G. lamblia in sewage using qPCR when applying the procedure for extraction of DNA from 1-liter sewage samples. Numbers obtained from the qPCR assay were comparable to those obtained with immunofluorescence microscopy. The qPCR analysis revealed both assemblage A and assemblage B genotypes of G. lamblia in the sewage. No Cryptosporidium was detected in these samples by either method.

* Corresponding author. Mailing address: Department of Biology, University of Toronto at Mississauga, 3359 Mississauga Rd. N, Mississauga, ON, Canada L5L 1C6. Phone: (905) 828-5430. Fax: (905) 828-3792. E-mail: rguy{at}utm.utoronto.ca.

Applied and Environmental Microbiology, September 2003, p. 5178-5185, Vol. 69, No. 9
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.9.5178-5185.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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