2.3. Cryptosporidium Genotyping and Subtyping
Fecal specimens preserved in potassium dichromate were washed three
times with distilled water by centrifugation at 2500×g for 5 min.
Genomic DNA was extracted from 0.2 g washed fecal material using the
FastDNA SPIN Kit for Soil (MP Biomedicals, CA, USA).Cryptosporidium species were detected using polymerase chain
reaction (PCR) and sequence analysis of the SSU rRNA gene. To identify
the C. parvum subtype involved, a ~850 bp
fragment of the gp60 gene was amplified using nested PCR
(Feng, Li, Duan, & Xiao, 2009).
Reagent-grade water was used as the negative control, whereas C.serpentis DNA was used as the positive control for the SSU rRNA
PCR and C. hominis DNA as the positive control for thegp60 PCR. All positive PCR products of the SSU rRNA andgp60 genes were sequenced bi-directionally on an ABI 3730 Genetic
Analyzer (Applied Biosystems, CA, USA). Nucleotide sequences
generated were aligned together
with reference sequences using ClustalX 2.1
(http://www.clustal.org/) for the
identification of Cryptosporidium species and C. parvumsubtypes.