Iranian J Parasitol: Vol. 6, No.4, 2011, pp.1-7 DNA extraction Total High Molecular weight DNA was ex- tracted by QIA amp DNA stool mini kit (Qiagen, Hilden, Germany). In according to manufacturer’s instructions, we performed a pre treatment as follows; 180 to 200 mg por- tion of each stool sample was transferred into an Eppendorf tube and dissolved in 700 µL of ASL buffer of DNA extraction kit. The samples were then exposed to a five cy- cles of freeze and thaw within liquid nitro- gen and boiling water in order to disruption of oocyst cells. Afterwards, 700 µL of ASL buffer was added into the sample tube and then the procedure was followed based on instruction of DNA extraction kit. Primer designing and Nested PCR amplification A part of 18S ribosomal RNA gene from Cryptosporidium genus which described previously (NCBI, Accession Nr. GQ259149.1) was selected to design specific primers for detecting of all Cryptosporidium species. The mentioned gene fragment was amplified with the primer pairs including: Cry18S-S2, 5’ GGTGACTCATAATAACTTTACGG 3’ as forward and Cry18S-As2, 5’ ACGCTATTGGAGCTGGAATTAC 3’ as reverse primers. Nested PCR was performed by the following primers; Cry18S-S1, 5’ TAAACGGTAGGGTATTGGCCT 3’ as forward and Cry18S-As1, 5’ CA- GACTTGCCCTCCAATTGATA 3’ as re- verse. PCR was carried out by using a peqS- TAR thermocycler (Peqlab, Germany) under the following conditions for both primary and Nested analysis: 94 °C for 5 min, 35 cy- cles of 1 min at 94 °C, 1.30 min at 60 °C, 2 min at 72 °C, followed by 35 cycles comprising 1 min at 94 °C, 1.30 min at 60 °C, 2 min at 72 °C and a final extension step of 10 min at 72 °C. 20 ρM of each primer was added in a volume of 50 µl containing: 20 mM (NH4)2 SO4, 75 mM 3 Tris-HCl (pH. 8.8), 1 mM MgCl2, 0.2 mM dNTP mix, 1.2 Units of thermo stable DNA polymerase (Roche), and 1 µl of template (genomic DNA). The amplification products were subjected to electrophoresis on a 1 % agarose gel. Result From 850 human samples and 100 cattle stool samples which have been investigated microscopically, 29 cases of cryptosporidio- sis infection in human and 30 positive sam- ples from cattle have been revealed. These positive samples were nominated for further PCR analysis. In addition, A total of 100 mi- croscopically negative human samples which, based on clinical manifestations, were suspected to cryptosporidiosis were selected for further PCR analysis. Isolation of DNA from positive feces sam- ples mentioned above has been done by DNA stool mini kit (Qiagen, Hilden, Ger- many). Synthesized Designed Primers from Crypto- sporidium 18S ribosomal RNA utilized in a double-tube nested PCR. Both outer and in- ner primers optimized at a same protocol. Using the outer primers, a fragment with ap- proximate size of 347 bp was amplified (Fig. 1), while by using inner primers a gene frag- ment with 240 bp has been produced (Fig. 2). The described primary and nested PCR method could detect all Cryptosporidium positive samples from human and cattle (Fig. 1&2). Regarding to suspected negative samples in primary PCR examination, the Nested PCR could approve two more positive results (Fig. 1&2). Furthermore, Nested PCR analysis was able to detect one more case which was negative in both microscopically examina- tion and primary PCR (Fig. 3). The specificity of the designed primers was approved by performing the same PCR