PCR and Real Time PCR for the Detection of Cryptosporidium parvum Oocyst DNA

Three DNA extraction kits were used, all without preliminary procedures, then DNA extraction was preceded with freeze/thaw cycles in three versions. A lack of desired effect resulted in the application of liquid nitrogen/water bath cycles before the use of the extractions in further experiments. The effectiveness of DNA extraction was measured by PCR signal and C(T) values of real time PCR. A comparison of the efficiency of various Cryptosporidium parvum undiluted oocyst treatments prior to DNA extraction with the use of three kits has shown that the best results were obtained after extraction of DNA with the Q1Aamp DNA Tissue Mini Kit (T kit), preceded by triple liquid nitrogen/water bath in 100 C for 2 minutes and with overnight proteinase K digestion. After extraction with the T kit, the detection limit was 50 oocysts per 200 mu l when effectiveness was evaluated with PCR and 10 oocysts in the case of real time PCR.