Detection and genotyping of oocysts of Cryptosporidium parvum by real-time PCR and melting curve analysis.
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 beta-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 beta-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.
Tanriverdi
Sultan
S
Department of Pediatric Hematology-Oncology, School of Medicine, Cukurova University, 01330 Adana, Turkey.
Tanyeli
Atila
A
Başlamişli
Fikri
F
Köksal
Fatih
F
Kilinç
Yurdanur
Y
Feng
Xiaochuan
X
Batzer
Glenda
G
Tzipori
Saul
S
Widmer
Giovanni
G
D000818:Animals; D052160:Benzothiazoles; D002417:Cattle; D003457:Cryptosporidiosis / Q000469:parasitology*; D016785:Cryptosporidium parvum / Q000145:classification* / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D003959:Diamines; D005456:Fluorescent Dyes; D005838:Genotype; D006801:Humans; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012680:Sensitivity and Specificity; D013696:Temperature; D014404:Tubulin / Q000235:genetics
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D014404:Tubulin; C098022:SYBR Green I