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From April 2000 to April 2001, three drinking-water–associated outbreaks of cryptosporidiosis occurred in Northern Ireland. These outbreaks were epidemiologically unrelated and originated from geographically separate areas. Concerns have been raised about a possible relationship between C. parvum genotypes and subgenotypes associated with these outbreaks. In this study, for genotyping analysis, we investigated these outbreaks using a small subunit rRNA (SSU rRNA)-based polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) genotyping tool, as well as the Cryptosporidium oocyst wall protein (COWP) PCR assay. For subgenotyping analysis, sequence typing of the 60-kDa glycoprotein (GP60) was used.

Genotypes were confirmed by using an SSU rRNA-based PCR-RFLP tool (12). Subgenotyping was done by sequence analysis of the GP60 gene (13). Before molecular analysis, the wastewater sample was processed by both salt flotation (11) and immunomagnetic separation (Dynal, Lake Success, NY), following the manufacturer-recommended procedures (14). Both genotyping and subgenotyping tools used nested PCR amplification of targeted genes. The primers used for GP60 were 5´-ATA GTC TCC GCT GTA TTC-3´ and 5´-TCC GCT GTA TTC TCA GCC-3´ for primary PCR and 5´-GGA AGG AAC GAT GTA TCT-3´ and 5´-GCA GAG GAA CCA GCA TC-3´ for secondary PCR. The PCR reaction contained 1X Perkin-Elmer (Norwalk, CN) PCR buffer, 3 mM MgCl2, 200 µM (each) deoxynucleoside triphosphate, 200 nM of the forward and reverse primers, 5 units of Taq polymerase, and 0.5–2 µL of DNA template (for primary PCR) or 2 µL of primary PCR product (for secondary PCR) in a total 100-µL reaction mixture. Each PCR reaction was then subjected to 35 cycles of denaturation at 94°C for 45 seconds, annealing at 50°C for 45 seconds, and extension at 72°C for 60 seconds, with an initial denaturation at 95°C for 3 minutes and a final extension at 72°C for 10 minutes. PCR products were sequenced in both directions on an ABI3100 (Applied Biosystems, Foster City, CA) with forward and reverse primers. An additional sequencing primer (5´-GAG ATA TAT CTT GGT GCG-3´) was used in the sequencing of GP60 PCR products. We aligned the study’s GP60 nucleotide sequences with each other and with sequences from the GenBank database with GCG software (Genetics Computing Group, Madison, WI). A neighbor-joining tree was constructed from the aligned sequences as described (15).

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