title: Validation of fragment analysis by capillary electrophoresis to resolve mixed infections by Cryptosporidium parvum subpopulations.
abstract: The potential of capillary electrophoresis (CE)-based DNA fragment analysis to identify mixed infections by Cryptosporidium parvum subpopulations was validated using high-resolution slab-gel electrophoresis. A selection of genomic DNA samples from C. parvum isolates with CE electropherogram profiles indicative of two concurrent alleles at one or more of six mini and microsatellite loci (MSB, MS5, ML1, ML2, TP14, 5B12) were analysed. These loci were PCR-amplified and products separated on precast Spreadex EL600 slab gels. ML1 PCR products differing by as little as 3 bp in length were visible after Spreadex gel electrophoresis and fragments were clearly separated for all but the ML2 and 5B12 loci, which generated stutter bands. No stuttering was seen for the remaining markers, having three or more nucleotide motifs in the repeat region. For each sample, the two bands of interest were excised separately, DNA extracted and re-amplified by PCR. Sequencing of these PCR products revealed the expected sequences for both alleles at most samples, except for the longest ML2 and 5B12 alleles which generated indeterminate sequences. Two novel MS5 alleles were successfully sequenced after PCR re-amplification. These findings demonstrate the utility of high-resolution Spreadex gels for analysing the polymorphism of satellite markers of Cryptosporidium isolates and support the validity of CE as a reliable and sensitive tool for detecting mixed Cryptosporidium subpopulations in a single-host infection.
authors.affiliation: Department of Animal Pathology, Faculty of Veterinary Sciences, University of Zaragoza, Miguel Servet 177, 50013, Zaragoza, Spain, jquilez@unizar.es.
authors.forename: Joaquín
authors.initials: J
authors.lastname: Quílez
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Hadfield
authors.forename: Ana
authors.initials: A
authors.lastname: Ramo
authors.forename: Claudia
authors.initials: C
authors.lastname: Vergara-Castiblanco
authors.forename: Rachel M
authors.initials: RM
authors.lastname: Chalmers
mesh_terms: D000483:Alleles; D060085:Coinfection; D003457:Cryptosporidiosis / Q000175:diagnosis / Q000469:parasitology*; D016785:Cryptosporidium parvum / Q000145:classification / Q000235:genetics*; D016054:DNA, Protozoan / Q000235:genetics; D019075:Electrophoresis, Capillary; D006801:Humans; D018895:Microsatellite Repeats; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D017422:Sequence Analysis, DNA
chemical_list: D016054:DNA, Protozoan