Cryptosporidium Genotyping by Nested PCR

Cryptosporidium species/genotypes were determined by nested PCR amplification of the small subunit ribosomal RNA (SSU rRNA) gene using primers F1 (5′-CCCATTTCCTTCGAAACAGGA-3′) and R1 (5′-TTCTAGAGCTAATACATGCG-3′) for primary amplification and F2 (5′-AAGGAGTAAGGAACAACCTCCA-3′) and R2 (5′-GGAAGGGTTGTATTTATTAGATAAAG-3′) for secondary amplification [16 , 17 (link)]. PCR reaction (25 μL) was composed of 1x PCR buffer (Mg²⁺-free), 2 mM MgCl₂, 200 μM of each deoxyribonucleoside triphosphate (dNTP), 0.4 μM of each primer, 0.2 U of HotStart Taq DNA polymerase (Takara, Dalian, China), and 2 μL of DNA template. The cycling conditions were 5 min at 95°C for initial denaturation and then 35 cycles of 45 s at 94°C, 45 s at 55°C, and 1 min at 72°C, followed by final extension at 72°C for 10 min. Both positive and negative controls were included in each test. PCR products were observed under UV light after electrophoresis in 1.5% agarose gel containing GoldView™ (Solarbio, Beijing, China).