AWWA PATH50781

This study examines the effect of pretreatment samples upon the recovery of DNA used for the template for polymerase chain reaction (PCR) and the relationship between the number of oocysts and the threshold cycle. The quantitation of oocysts was based on the use of dual-labeled fluorogenicprobe and the detection of fluorescent light intensity (measured as CT values). The oocysts used in this study were recovered from feces of a two week old calf and were purified by the immunomagnetic separation method. PCR was performed with the use of commercially available reagents. Two reported primers and two newly designed primers were used. In this experiment, samples treated with both alkali and protenase K showed the lowest CT value. With regard to the sensitivity, as low as one oocyst was detected by this real time quantitative PCR method. A good correlation between the number of oocysts and Ct values was observed. The total time required to perform this test was approximately 6.5 hours (3.5 hours for pretreatment and 3 hours for PCR). Includes 4 references, tables, figures.