|Lindquist, H.d. alan|
|Schaefer, iii, Frank|
Submitted to: Applied and Environmental Microbiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 5/29/2009
Publication Date: 6/1/2009
Publication URL: hdl.handle.net/10113/30765
Citation: Yang, W., Lindquist, H., Cama, V., Schaefer, Iii, F.W., Villegas, E., Fayer, R., Lewis, J., Xiao, L. 2009. Detection of Toxoplasma gondii oocysts in water sample concentrates by real-time PCR. Applied and Environmental Microbiology. 75(11):3477-83. Interpretive Summary: Toxoplasma gondii is a protozoan parasite infectious for virtually all vertebrates. Cats are the only animals capable of excreting the oocyst stage into the environment and contaminating surface waters with the infectious form of the parasite. The present study was devised to find a rapid and precise method to detect this form of the parasite in surface waters. Using specialized filters, that can remove microscopic particles from at least 100 liters of water, combined with molecular methods referred to as real-time PCR, as little as one oocyst could be detected within a pellet of debris extracted from surface water using the specialized filter. This method can be utilized by water treatment plants, agencies and companies examining the safety of drinking water, and for epidemiological tracing following outbreaks suspected to be linked to contaminated water.
Technical Abstract: PCR techniques in combination with conventional parasite concentration procedures have potential for 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 compared for detection of T. gondii tachyzoites and oocysts. Lower sensitivity and specificity were obtained using the B1 gene-based PCR than by the 529-bp repeat-based PCR. New procedures for real-time PCR detection of T. gondii oocysts in concentrates of surface water were developed and tested in conjunction with a method for 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.