Cryptosporidium genotyping

Zhao, Wei, Zhou, Huanhuan, Huang, Yi, Xu, Liulian, Rao, Langyu, Wang, Shanshan, Wang, Wenqi, Yi, Yufang, Zhou, Xiaojun, Wu, Yue, Ma, Tianming, Wang, Gaoyu, Hu, Xiaoyuan, Peng, Ruoyan, Yin, Feifei & Lu, Gang, 2019, Cryptosporidium spp. in wild rats (Rattus spp.) from the Hainan Province, China: Molecular detection, species / genotype identification and implications for public health, International Journal for Parasitology: Parasites and Wildlife 9, pp. 317-321 : 318-319

2.5. Cryptosporidium genotyping and subtyping

All DNA preparations were tested for the presence of Cryptosporidium spp. by nested PCR amplification of an 830-bp nucleotide fragment of the SSU rRNA gene as previously described ( Xiao et al., 1999). All PCR amplifications were performed with positive controls ( C. homini DNA for Cryptosporidium ) and negative controls (2μ deionized water) which contained no DNA. Subtyping of C. viatorum samples was performed through nested PCR amplification of ∼800–850-bp fragments of the gp60 gene as described by Stensvold et al. (2015). TaKaRa TaqDNA Polymerase (TaKaRa Bio Inc., Tokyo, Japan) was used for all PCR reactions. All secondary PCR products were subjected to electrophoresis on 1.5% agarose gels and visualized by DNAGREEN staining (Tiandz, Inc., Beijing, China).

2.6. DNA sequencing and analysis

All secondary PCR products were sequenced using the same secondary PCR primers on an ABI PRISM™ 3730 DNA Analyser (Applied Biosystems, Carlsbad, CA, USA) using a BigDye Terminator v3·1 Cycle Sequencing kit (Applied Biosystems). The accuracy of the sequencing data were confirmed by sequencing the PCR products in both directions. Further PCR products from specific DNA preparations were sequenced as required. Nucleotide sequences obtained in the present study were subjected to BLAST searches (http://www.ncbi.nlm.nih. gov/blast/) and then analyzed and aligned with each other and the published reference sequences of Cryptosporidium in GenBank, using ClustalX 1.81 (http://www.clustal.org/).