Mboii

Genomic DNA was extracted from 200 μl EDTA-anticoagulated peripheral blood sample with a DNA isolation kit (BioTeke, Peking, China) as the manufacturer's direction. DNA was stably stored at −20°C until assayed. Genotyping of the IL-31 gene polymorphism was conducted by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). We designed the PCR primers with software Primer 3 (http://bioinfo.ut.ee/primer3‐0.4.0/primer3/) [27 (link)] as shown in Table 1. The 10 μl PCR reaction system was consisted of 1.0 μl DNA and 5 μl 2× Power Taq PCR Master Mix (BioTeke, Peking, China), forward and reverse primer 0.1 μl, respectively, and reserved volume was made up to 10 μl by sterilized water. The PCR condition was designed as 95°C for 4 min firstly, then 33 cycles at 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s, and finally, 72°C for 10 min. Furthermore, the PCR products were digested in 37°C stable incubation by distinguished restriction enzyme MboII (New England Biolabs, Peking, China) for 30 minutes of rs4758680 and ScrFI (New England Biolabs, Peking, China) for 2 hours of rs7977932 as shown in Table 1, separately. Ultimately, the results were visually analyzed by 6% polyacrylamide gels in silver staining. To verify the genotyping results, DNA sequencing was performed in about 20% PCR-amplified DNA samples randomly.

The secondary PCR products of the 18S rRNA gene were purified using the QIAquick PCR purification kit (QIAGEN) according to the manufacturer's instruction and were digested using the SspI or MboII (New England Bio Labs Inc.) restriction enzymes (Xiao et al., 1999 (link), Xiao et al., 2001 (link); Feng et al., 2007 (link)). Briefly, 10 μl of the purified secondary PCR product was digested with 5 units of enzyme and 2 μl of the corresponding 10× buffer in a final volume of 20 μl. All restriction digestions were carried out at 37 °C overnight, fractionated on 2% agarose gel and visualised after Gel red staining.

Nucleic acid was extracted from all fecal specimens and water filters using the FastDNA® SPIN Kit for Soil (MP Biomedicals, LLC, Solon, OH) following the methods described [20 (link)]. DNA extracts were subsequently tested using a polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) approach where a segment (∼833 bp) of the Cryptosporidium SSU rRNA gene is amplified by nested PCR and then species and genotype diagnosis is made by restriction digestion of the secondary PCR product with SspI (New England BioLabs, Beverly, MA), and either VspI (Promega, Madison, WI) or MboII (New England BioLabs) [21 (link),22 ]. Each sample was run in duplicate by PCR-RFLP analyses with appropriate controls. Specimens that were positive for Cryptosporidium by the SSU rRNA PCR were confirmed by DNA sequencing of the 18S PCR products (C. suis, C. xiaoi and C. hominis). A subset of specimens positive for C. hominis were also subtyped by sequencing the 60-kilodalton glycoprotein (GP60; ∼900 bp) in both directions on an ABI 3130 Genetic Analyzer (Foster City, CA) [23 (link)]. All sequences obtained were aligned with reference sequences using MEGA 6.0 or ClustalX software (http://www.clustal.org/) to identify Cryptosporidium species and C. hominis genotypes.