Host-adapted Cryptosporidium and Enterocytozoon bieneusi genotypes in straw-colored fruit bats in Nigeria

Li, Na; Ayinmode, Adekunle B.; Zhang, Hongwei; Feng, Yaoyu; Xiao, Lihua

2.2. Detection of Cryptosporidium spp., G. duodenalis and E. bieneusi DNA was extracted from 200 μl of stored fecal specimens using the FastDNA SPIN Kit for Soil (MP Biomedicals, Santa Ana, CA). This technique was shown to be better in removing PCR inhibitors in environmental samples than other common commercial DNA extraction kits (Jiang et al., 2005) . The extracted DNA was stored at −80°C before analysis by PCR. To detect Cryptosporidium spp., a ∼830-bp fragment of the small subunit (SSU) rRNA gene was amplified by nested PCR, and Cryptosporidium genotypes were initially identified by restriction fragment length polymorphism (RFLP) analysis of the secondary PCR products using restriction enzymes SspI and VspI (New England BioLabs, Massachusetts, USA) (Xiao et al., 1999) . To detect G. duodenalis, a ∼530-bp fragment of the triosephosphate isomerase (tpi) gene was amplified by nested PCR (Sulaiman et al., 2003a) . To detect E. bieneusi, a 392-bp fragment of the rRNA unit containing the entire internal transcribed spacer (ITS) was amplified by nested PCR (Sulaiman et al., 2003b) . Each specimen was analyzed by PCR twice using 1 μl of extracted DNA per PCR, with DNA from C. canis as the positive control for the SSU rRNA PCR, DNA from G. duodenalis assemblage C as the positive control for the tpi PCR, and DNA from E. bieneusi genotype PtEb IX as the positive control for the ITS PCR. Two negative controls (reagent-grade water) for primary PCR and secondary PCR were further used in each PCR run. Non-acetylated bovine serum albumin (Sigma-Aldrich, St, Louis, MO, USA) was used at the concentration of 400 ng/μl in the primary PCR to neutralize residual PCR inhibitors in DNA, as previously described (Jiang et al., 2005) .