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2019, Cilt 49, Sayı 3, Sayfa(lar) 162-168
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Method Verification Study for Detection of Cryptosporidium spp. by Realtime PCR Method
Selma Usluca1, Asiye Evren Eken Berberoğlu1, Bekir Çelebi2, Selçuk Kılıç1
1Halk Sağlığı Genel Müdürlüğü, Mikrobiyoloji Referans Laboratuvarları ve Biyolojik Ürünler Daire Başkanlığı, Ankara
2Halk Sağlığı Genel Müdürlüğü, Zoonotik ve Vektörel Hastalıklar Daire Başkanlığı, Ankara
Keywords: Cryptosporidium spp., realtime PCR, method verification

Objective: Cryptosporidium spp. is an important parasite, especially among the causes of foodborne and waterborne diarrhea. The aim of this study was to verify the molecular method used for routine diagnosis of Cryptosporidium spp. in stool samples.

Methods: For this purpose, Cryptosporidium parvum positive calf stool sample was diluted with PBS and oocysts were counted using Cryptosporidium/Giardia direct fluorescein antibody assay. DNA extraction from the same sample was carried out using a commercial kit after determining the number of oocysts in the sample. PCR amplification was performed using multiplex PCR assay for molecular detection of Cryptosporidium spp., Giardia intestinalis, Entamoeba histolytica, Dientamoeba fragilis in the DNA sample retrieved.

Results: The detection limit was determined as 8 oocysts/reaction. Then, high and low positive samples were prepared, and accuracy and precision studies of the method were performed by two different analysts to complete verification analysis of the test method.

Conclusion: Since variation coefficients of method verification studies were below 15%, it was decided that the test was suitable for investigation of Cryptosporidium spp. in routine patient samples.


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