Molecular Detection of Cryptosporidium Subtypes

Cryptosporidium was detected by nested PCR amplification of the SSU rRNA gene fragment of ~830 bp. Primers and cycle parameters were designed by Xiao and colleagues (26 (link)). A fragment of ~948 bp of the 60-kDa glycoprotein (gp60) gene was used to identify C. ubiquitum subtypes via nested PCR amplification using the primers described by Li et al. (27 (link)). TaKaRa Taq DNA polymerase (TaKaRa Bio Inc., Tokyo, Japan) was used for all PCRs. PCR amplifications were performed with positive (chicken-derived C. bailey DNA) and negative controls (no DNA water). PCR products were visualized on a UV transilluminator following electrophoresis on 1.5% agarose gels stained with GelStrain (Trans Gen Biotech, Beijing, China).