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Applied and Environmental Microbiology, June 2009, p. 3477-3483, Vol. 75, No. 11
0099-2240/09/$08.00+0     doi:10.1128/AEM.00285-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Detection of Toxoplasma gondii Oocysts in Water Sample Concentrates by Real-Time PCR{triangledown}

Wenli Yang,1,2 H. D. Alan Lindquist,3 Vitaliano Cama,1 Frank W. Schaefer III,3 Eric Villegas,4 Ronald Fayer,5 Earl J. Lewis,6 Yaoyu Feng,7 and Lihua Xiao1*

Division of Parasitic Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30341,1 Atlanta Research and Education Foundation, 1670 Clairmont Road, Decatur, GA 30033,2 National Homeland Security Research Center, United States Environmental Protection Agency, Cincinnati, Ohio 45268,3 National Exposure Research Laboratory, United States Environmental Protection Agency, Cincinnati, Ohio 45268,4 Agricultural Research Service, United States Department of Agriculture, Beltsville, Maryland 20705,5 Center for Coastal Environmental Health and Biomolecular Research, National Oceanic and Atmospheric Administration, Oxford, Maryland 21654,6 School of Resource and Environmental Engineering, East China University of Science and Technology, Shanghai 200237, China7

Received 4 February 2009/ Accepted 31 March 2009

PCR techniques in combination with conventional parasite concentration procedures have potential for the sensitive and specific detection of Toxoplasma gondii oocysts in water. Three real-time PCR assays based on the B1 gene and a 529-bp repetitive element were analyzed for the detection of T. gondii tachyzoites and oocysts. Lower sensitivity and specificity were obtained with the B1 gene-based PCR than with the 529-bp repeat-based PCR. New procedures for the real-time PCR detection of T. gondii oocysts in concentrates of surface water were developed and tested in conjunction with a method for the direct extraction of inhibitor-free DNA from water. This technique detected as few as one oocyst seeded to 0.5 ml of packed pellets from water samples concentrated by Envirocheck filters. Thus, this real-time PCR may provide a detection method alternative to the traditional mouse assay and microscopy.

* Corresponding author. Mailing address: Division of Parasitic Diseases, National Center for Zoonotic, Vector-Borne and Enteric Diseases, Centers for Disease Control and Prevention, Building 22, Mail Stop F-12, 4770 Buford Highway, Atlanta, GA 30341. Phone: (770) 488-4840. Fax: (770) 488-4454. E-mail: lxiao{at}cdc.gov

{triangledown} Published ahead of print on 10 April 2009.

Applied and Environmental Microbiology, June 2009, p. 3477-3483, Vol. 75, No. 11
0099-2240/09/$08.00+0     doi:10.1128/AEM.00285-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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