Bairami et al. : A Sensitive and Specific PCR Based Method … Introduction Cprotozoan parasites causing water- ryptosporidium sp. are monoxenous food borne gastrointestinal infections in both human and animal (1-3). They are common seen in childhood, pregnancy and immune-compromised people such as AIDS patients (4). Children are the most infected group in developing countries (5-7). Cryptosporidium oocysts were detected in 13% of parsitologic stool investigations in developing countries (5-7). Species which involve human are C. muris, C. parvum, C. hominis, C. felis and C. canis. Oocysts contaminating food or water and direct contact with infected animals or humans cause acute gastroenteritis and diarrhea in healthy people but in immunocompromised patients, individuals with AIDS and malnourished children, Cryptosporidium parasites cause a chronic and life-threatening disease (1, 8, 9). Common methods for detection of Crypto- sporidium are parasite visualization using acid-fast staining as well as fluorescent staining after concentration. Because they are obligate intracellular parasites, cultivat- ing of the organism is not routine in the laboratory (10, 11). To obtain high sensitivity in the diagnosis with microscope, a modified Ziehl- Neelsen staining and a minimum amount of 500,000 oocysts in each gram of examined stool re- quired (11). Besides, identification of oo- cysts in direct microscopic detection is de- pended to the time as well as experience of stool examiner (12,13). In addition, the lack of morphological characters to discriminate Cryptosporidium species causes low sensitivity and specificity for detection of this parasite (3,4). Furthermore, Immunofluorescent-antibody assays (IFA) methods which used to detect Cryptosporid- ium oocysts in environmental samples are 2 not useful for species identification (14, 15- 17). The PCR techniques have proved both spe- cific and sensitive methods for detection of protozoan infections and Cryptosporidium specimen types (18, 19). In the present study, based on a primer designed from 18S ribo- somal RNA, we tried to set up a sensitive Nested-PCR for detection of Cryptosporid- ium species from human, and cattle feces. This method may help us for detection of these parasites in a more rapid and sensitive way. Materials and Methods Fecal specimens A total of 850 fecal samples were obtained from patients clinically suspected to crypto- sporidiosis. One hundred stool specimens from diarrheic and/or healthy cattle were also collected from rural area in south of Iran. The samples were referred to Parasitol- ogy Laboratory, School of Public Health, Tehran University of Medical Sciences the simplified Formol-Ether concentration me- thod was carried out for all samples (20, 21). They were then examined and evaluated microscopically by modified Ziehl-Neelsen staining method. Briefly, thin smears of fe- cal suspension were prepared on glass slides. The slides were flooded with carbol fuchsin for 1 hour following fixing by absolute alco- hol (20). Then they were washed and decolorized in 3% acid-alcohol for about 30 seconds. The slides were then washed and stained with 1% methylene blue for 4 mi- nutes. After washing and air drying, the slides were investigated microscopically by 40× as well as 100× objectives (20). Positive samples were separated for performing PCR analysis.