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Detection and Genotyping of Oocysts of Cryptosporidium parvum by Real-Time PCR and Melting Curve Analysis

Sultan Tanriverdi,¹ Atila Tanyeli,¹ Fikri Balamili,² Fatih Köksal,³ Yurdanur Kilinç,¹ Xiaochuan Feng,⁴ Glenda Batzer,⁴ Saul Tzipori,⁴ and Giovanni Widmer^1*

Department of Pediatric Hematology-Oncology,¹ Department of Hematology-Oncology,² Department of Microbiology, School of Medicine, Çukurova University, 01330 Adana, Turkey,³ Division of Infectious Diseases, Tufts University School of Veterinary Medicine, North Grafton, Massachusetts 01536⁴

Received 10 April 2002/ Returned for modification 29 May 2002/ Accepted 25 June 2002

Several real-time PCR procedures for the detection and genotyping of oocysts of Cryptosporidium parvum were evaluated. A 40-cycle amplification of a 157-bp fragment from the C. parvum ß-tubulin gene detected individual oocysts which were introduced into the reaction mixture by micromanipulation. SYBR Green I melting curve analysis was used to confirm the specificity of the method when DNA extracted from fecal samples spiked with oocysts was analyzed. Because C. parvum isolates infecting humans comprise two distinct genotypes, designated type 1 and type 2, real-time PCR methods for discriminating C. parvum genotypes were developed. The first method used the same ß-tubulin amplification primers and two fluorescently labeled antisense oligonucleotide probes spanning a 49-bp polymorphic sequence diagnostic for C. parvum type 1 and type 2. The second genotyping method used SYBR Green I fluorescence and targeted a polymorphic coding region within the GP900/poly(T) gene. Both methods discriminated between type 1 and type 2 C. parvum on the basis of melting curve analysis. To our knowledge, this is the first report describing the application of melting curve analysis for genotyping of C. parvum oocysts.

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