Fecal aliquots without any additive were subjected to PCR test within 2 weeks from storage. DNA samples were isolated and purified using QIAamp® Stool Mini Kit (Qiagen, Hilden, Germany). The extraction procedure was carried out following the changes we have introduced over amended kit's protocol in a previous study [14 (link)]. A specific DNA sequence of the Cryptosporidium oocyst wall protein (COWP) gene was amplified using previously published primers [16 (link)]. Amplification reactions were done using Techne™ TC-4000 thermal cycler. The GoTaq® Hot Start Polymerase (Promega, Heidelberg, Germany) and other reagents were used in PCR with final concentrations closely similar to the published protocol. PCR products were analyzed on 1-2% of agarose gel electrophoresis.
Protocol detail
Fecal DNA Extraction and PCR Detection of Cryptosporidium
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A gene
Agarose gel electrophoresis
Cryptosporidium oocyst wall protein
Dna sequence
Primers
Promega
Stool
Corresponding Organization :
Other organizations : Taif University, Menoufia University
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Variable analysis
independent variables
- Fecal aliquots without any additive
- Presence/absence of Cryptosporidium oocyst wall protein (COWP) gene
- Storage time (within 2 weeks)
- DNA extraction procedure (QIAamp® Stool Mini Kit with introduced changes)
- Thermal cycler (Techne™ TC-4000)
- PCR reagents (GoTaq® Hot Start Polymerase and other reagents used with similar concentrations to published protocol)
- Agarose gel electrophoresis (1-2% gel)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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