Computational protocol: Molecular Characterization of Cryptosporidium spp. among Children in Rural Ghana

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Protocol publication

[…] Immediately after stool collection, the sample was frozen at −20°C. DNA was extracted from the frozen stool samples using the QIAamp DNA Stool-Kit (Qiagen, Hilden, Germany). Extracted DNA was transported to Germany and subsequently a real-time polymerase chain reaction (RT-PCR) test was applied to identify C. hominis/C. parvum DNA as previously described []. The PCR targets the 18S small subunit ribosomal RNA gene and amplifies a 139 bp DNA fragment. As controls for DNA extraction and/or PCR inhibition each sample was contaminated with traces of phocine Herpesvirus-1 (PhHV-1) prior to faecal DNA extraction. RT-PCR for PhHV-1 was performed as previously described []. All PCR-confirmed Cryptosporidium spp. DNA samples were subject to amplification of a 850-bp fragment of the gp60 gene using a nested PCR with the outer primers AL3531 and AL3535 and inner primers AL3532 and AL3534 as described before [,]. Amplicons were sent for purification and bidirectional sequencing to Eurofins (Hamburg, Germany). Forward and reverse sequences were assembled using Seqscape Software v3.0 (Applied Biosystems, USA) and subsequently aligned using the ClustalW program running within the BioEdit software package, version Classification on species level was conducted by an alignment to reference sequences retrieved from the NCBI GenBank. The assignment to subtypes was performed according to the number of trinucleotide repeats (TCA, TCG or TCT) coding for the amino acid serine as described elsewhere []. The subtype IIcA5G3 was subdivided according to differences in the 3’ region of the gp60 gene []. To further support the subtype classification, a neighbour-joining tree was built using the Kimura two-parameter model in MEGA 5 []. The reliability of all phylogenetic groupings was determined through a bootstrap resampling analysis (1,000 replicates). Six reference sequences of Cryptosporidium spp. subtypes from the NCBI GenBank database were included in the alignment as controls.All sequences were uploaded to NCBI GenBank (Accession numbers KM538975—KM539062). […]

Pipeline specifications

Software tools SeqScape, Clustal W, BioEdit, MEGA
Applications Phylogenetics, Sanger sequencing
Organisms Cryptosporidium parvum, Homo sapiens, Campylobacter hominis
Diseases Gastrointestinal Diseases, Infection