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.