Iranian J Parasitol: Vol. 6, No.4, 2011, pp.1-7 Fig. 3: PCR & nested-PCR products of a human sample that was negative in both microscopi- cally and primary PCR analysis on 1% agarose gel. WM: 100 bp molecular weight marker; lane 1: sample k PCR pattern; lane 2: sample k nested-PCR pattern Discussion We described a sensitive and specific PCR based method for the rapid and direct detec- tion of Cryptosporidium in stool specimens. Cryptosporidiosis, as mentioned before, is one of the most important emerging infec- tious diseases which are directly transmitted by the fecal–oral route and cause different kinds of diseases in mammals, birds, and fish (22). Although current laboratory meth- ods for the detection of Cryptosporidium oocysts are primarily relied on examination by microscopy, however, morphological characters for identifying Cryptosporidium are few, and identification based on light mi- croscopy alone is unreliable and relatively time consuming (23, 24). In addition, re- searches shown that methods based on immunological detections are not more sensitive than conventional microscopy (13, 25). In contrast, because of sensitivity and easily performance, PCR amplification seems to be an obvious choice for improved detection of Cryptosporidium from feces. To the best of our knowledge, PCR-based as- says have not been previously developed lo- 5 cally in Iran for direct identification of Cryptosporidium genus. However, some investigations have been performed in order to differentiation the related species (26-28). In this study, we designed novel outer and inner primer pairs with a same annealing temperature in both analyses. Our investiga- tion showed the ability of the first PCR with the outer 18S rRNA primers in detection of Cryptosporidium infected samples. In addi- tion, in some cases with small amount of parasite, in which the direct microscopy as well as primary PCR were unable to detect the infection, the nested-PCR with inner 18S rRNA primers was able to recognize the infection. The study of Weber et al, revealed that the sensitivity of Immunofluorescence (IF) diagnostic methods for detecting Cryptosporidium oocysts in human stools was related to the amount of oocysts. The minimum of amounts of oocysts per gram were indicated as 10,000 in watery stool, 50,000 in formed stools (11). In addition, at least 500,000 oocysts per gram of formed stool were needed for a 100% detection rate