title: Whole genome amplification (WGA) for archiving and genotyping of clinical isolates of Cryptosporidium species.
abstract: Clinical and environmental isolates of pathogens are often unique and may be unculturable, yielding a very limited amount of DNA for genetic studies. Cryptosporidium in particular are difficult to propagate. Whole genome amplification (WGA) is a valuable technique for amplifying genomic material. In this study, we tested 5 WGA commercial kits using Cryptosporidium clinical isolates. DNA of 5 C. hominis and 5 C. parvum clinical isolates and C. parvum IOWA reference strain were used. The majority of the samples were amplified by all of the kits tested. The integrity and fidelity of the amplified genomic DNA were assessed by sequence analysis of several PCR products of varying length. We found evidence that one kit in particular may be more error prone while another seemed the more suitable kit for Cryptosporidium clinical samples, generating high molecular weight DNA from all the samples with high fidelity. Thus WGA was found to be a useful technique for producing amplified DNA suitable for downstream genotyping techniques and archiving of Cryptosporidium clinical isolates.
authors.affiliation: Biomedical Research Centre, School of Medicine, Health Policy and Practice, University of East Anglia, Norwich NR4 7TJ, UK.
authors.forename: Maha
authors.initials: M
authors.lastname: Bouzid
authors.forename: Darren
authors.initials: D
authors.lastname: Heavens
authors.forename: Kristin
authors.initials: K
authors.lastname: Elwin
authors.forename: Rachel M
authors.initials: RM
authors.lastname: Chalmers
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Hadfield
authors.forename: Paul R
authors.initials: PR
authors.lastname: Hunter
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Tyler
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000818:Animals; D002648:Child; D002675:Child, Preschool; D003457:Cryptosporidiosis / Q000453:epidemiology / Q000469:parasitology; D003458:Cryptosporidium / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D016785:Cryptosporidium parvum / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D016054:DNA, Protozoan / Q000032:analysis*; D005260:Female; D018503:Genome, Protozoan; D005838:Genotype; D006801:Humans; D008297:Male; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D013045:Species Specificity; D055815:Young Adult
chemical_list: D016054:DNA, Protozoan; D011933:Reagent Kits, Diagnostic