Cryptosporidium genotyping

Wei, Zilin, Liu, Qiang, Zhao, Wei, Jiang, Xiumei, Zhang, Ying, Zhao, Aiyun, Jing, Bo, Lu, Gang & Qi, Meng, 2019, Prevalence and diversity of Cryptosporidium spp. in bamboo rats (Rhizomys sinensis) in South Central China, International Journal for Parasitology: Parasites and Wildlife 9, pp. 312-316 : 313

2.4. Cryptosporidium genotyping and subtyping

All genomic DNA samples were subjected to nested PCR targeting Cryptosporidium by amplification of an 830 bp nucleotide fragment of the small subunit (SSU) rRNA of Cryptosporidium . Primers were designed as previously described ( Xiao et al., 1999). C. parvum was further subtyped by nested PCR amplification of an 800–850 bp fragment of the 60-kDa glycoprotein (gp60) ( Alves et al., 2003). All the isolates of Cryptosporidium -positive samples were selected for further sequence characterization at the 70-kDa heat shock protein (HSP70) gene, oocyst wall protein (COWP) gene and actin gene ( Sulaiman et al., 2000, 2002; Xiao et al., 2000). PCR mixtures (25 μL total volume) were prepared for amplification of the SSU rRNA, HSP70, COWP and gp60 genes. PCRs consisted of 1 μL of genomic DNA for primary PCRs, and 1 μL of amplification product for secondary PCRs, 12.5 μL of 2 × Easy Taq PCR SuperMix (Trans Gene Biotech Co. Ltd., Beijing, China), 0.3 μM forward and reverse primers, and 10.9 μL of deionized water. All PCRs included positive controls (chicken-derived C. bailey DNA ) and negative controls (containing no template DNA). Secondary PCR products were visualized on 1.5% agarosegels stained with GelRed (Biotium Inc., Hayward, CA, USA) prior to sequencing.