Infections caused by Toxoplasma gondii are widely prevalent in animals and humans throughout the world. In the United States, an estimated 23% of adolescents and adults have laboratory evidence of infection with T. gondii. T. gondii has been identified as a major opportunistic pathogen in immunocompromised individuals, in whom it can cause life-threatening disease. Water contaminated with feces from domestic cats or other felids may be an important source of human exposure to T. gondii oocysts. Because of the lack of information regarding the prevalence of T. gondii in surface waters, there is a clear need for a rapid, sensitive method to detect T. gondii from water. Currently available animal models and cell culture methods are time consuming, expensive, and labor-intensive, requiring days to weeks for results to be obtained. Detection of T. gondii nucleic acid by the polymerase chain reaction (PCR) has become the preferred method. We have developed a PCR amplification and detection method for T. gondii oocyst nucleic acid that incorporates the use of hot start amplification to reduce non specific primer annealing, Uracil-N-glycosylase to prevent false-positive results due to carryover contamination, an internal standard control to identify false-negative results due to inadequate removal of sample inhibition, and PCR product oligoprobe confirmation using a non-radioactive DNA hybridization immunoassay. This method can provide positive, confirmed results in less than one day. Using cloned T. gondii DNA, detection sensitivity is less than 200 femtograms. Less than 100 oocysts can be detected following recovery of oocyst DNA. Development of a T. gondii oocyst PCR detection method will provide a useful technique to estimate the levels of T. gondii oocysts present in surface waters.
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