Cryptosporidium Genotyping Techniques. CryptoNet has been built for comparison of the 18S rRNA genes and 90 kDa glycoprotein gene (gp60) and has established a DNA sequence database using:
- A nested PCR-RFLP method on the 18S rRNA gene extracted from cryptosporidiosis diagnostic samples. The 18S rRNA gene has high copy numbers and the presence of conserved regions interspersed with highly polymorphic regions. The 800 bp nested PCR amplicon is used in RFLP reactions with different sets of restriction enzymes to differentiate Cryptosporidium species and genotypes.
- A nested PCR method on the gp60 gene extracted from clinical samples submitted for subtyping of Cryptosporidium species. GP60 is the most commonly used target for subtyping C. parvum, C. hominis, and C. meleagridis. Sequencing of the 800 bp nested PCR product identifies subtype family and subtype.
Laboratory Resource Requirements and Costs. Laboratories that regularly perform molecular analysis will need little to no additional equipment or resources to complete the necessary methodologies for Cryptosporidium genotyping. Specific equipment needs include a thermal cycler, microcentrifuge, electrophoresis system, water bath, PCR workstation, ABI 3130xl Genetic Analyzer or similar sequencing machine, and BioNumerics software. The estimated cost per sample beginning with DNA isolation and ending with DNA sequencing is approximately $9.50 1. CDC will cover expenses to train and certify partnering laboratories across the U.S.
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- Cost may vary depending on the brand of reagents and consumables, as well as contract pricing.