Iranian J Parasitol: Vol. 6, No.4, 2011, pp.1-7 Iranian J Parasitol Open access Journal at http:// ijpa.tums.ac.ir Iranian Society of Parasitology http:// isp.tums.ac.ir Tehran University of Medical Sciences Publication http:// tums.ac.ir Original Article A Sensitive and Specific PCR Based Method for Identification of Cryptosporidium Sp. Using New Primers from 18S Ribosomal RNA A Bairami Kuzehkanan1, 2, M Rezaeian1,3, H Zeraati4, M Mohebali1,3, AR Meamar 5, Z Babaei6, L Kashi1, M Heydarnezhadi1, *S Rezaie1,7 1Dept. of Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2Dept. of Public Health, School of Public Health, Alborz University of Medical Sciences, Alborz, Iran 3Center for Research of Endemic Parasites of Iran (CREPI), Tehran University of Medical Sciences, Tehran, Iran 4Epidemiology and Biostatistics Department, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 5Dept. of Parasitology and Mycology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran 6Dept. of Parasitology and Mycology, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran 7Dept. of Medical Biotechnology, School of Advanced Medical Sciences, Tehran University of Medical Sciences, Tehran, Iran (Received 11 Apr 2011; accepted 27 Oct 2011) Abstract Background: The main goal of the present study was to develop a new sensitive and specific PCR based method for Identification of Cryptosporidium sp. using novel primers from 18S ribosomal RNA. Cryptosporidiosis in high-risk host groups particularly in neonates and immuno-compromised individuals may result in death. To the best of our knowledge this is the first study regarding develop a new PCR based method to diagnose the cryptosporidiosis in Iran. Methods: A total of 850 human fecal samples from patients clinically suspected to cryptosporidiosis and 100 healthy and diarrheic cattle stool specimens were collected. The simplified formol-ether concentration method was carried out for all samples. They were then examined microscopically by modified Ziehl-Neelsen staining method. Total DNA was extracted by QIA amp DNA stool mini kit. PCR and nested-PCR was carried out by using designed prim- ers. Results: Twenty nine cases of cryptosporidiosis infection in human and 30 samples from cattle microscopically were positive. The described primary and nested PCR method could detect all Cryptosporidium positive samples from human and cattle. Regards to suspected negative samples in primary PCR examination, the Nested PCR could ap- prove two more positive results. Furthermore, Nested PCR analysis was able to detect one more case which was negative in both microscopically examination and primary PCR. Specificity of the test was 100%. Sensitivity of Nested PCR in comparison to our gold standard; microscopy after Ridley concentration modified ziehl-Neelsen, was 100 %. Conclusion: Our developed PCR based method by using new primers devised from 18S ribosomal RNA revealed the ability for identification of the Cryptosporidium species such as C. parvum and C. huminis with high specificity and sensitivity. Keywords: Cryptosporidium, PCR, Identification *Corresponding author: Tel: +98 912 1218439, Fax: +98 21 88951392, E-mail: srezaie@sina.tums.ac.ir. 1