Capillary electrophoretic analysis of fragment length polymorphism in ribosomal markers of Cryptosporidium from humans

• Published in: Molecular and cellular probes 2005-12, Vol.19 (6), p.394-399
• Main Authors: Schindler, A. Regina, Abs EL-Osta, Youssef G., Stevens, Melita, Sinclair, Martha I., Gasser, Robin B.
• Format: Article
• Language: English
• Subjects: Animals; Capillary electrophoresis; Cost-Benefit Analysis; Cryptosporidium - genetics; Cryptosporidium hominis; Cryptosporidium parvum; Cryptosporidium parvum - genetics; Diagnosis; DNA, Ribosomal - analysis; DNA, Ribosomal Spacer - analysis; DNA, Ribosomal Spacer - chemistry; Electrophoresis, Capillary - methods; Genetic Variation; Human cryptosporidiosis; Humans; Polymorphism, Restriction Fragment Length; Ribosomal DNA; Sequence Analysis, DNA - methods; Specific identification
• ISSN: 0890-8508; 1096-1194
• DOI: 10.1016/j.mcp.2005.07.001

Cryptosporidium oocyst DNA samples ( n=80) from humans with cryptosporidiosis in Australia and the UK were characterized genetically and categorized by capillary electrophoretic (CE) analysis of part of the small subunit gene (pSSU; ∼300 bp) and second internal transcribed spacer (pITS-2; ∼230 bp) of nuclear ribosomal DNA. The amplicons were heat denatured and subjected to capillary electrophoresis in LPA matrix (Amersham) in a MegaBACE™ 1000 system (Amersham). The chromatograms captured were stored electronically and then analysed using MegaBACE™ Fragment Profiler software. Using reference DNA control samples representing Cryptosporidium hominis and Cryptosporidium parvum, particular peaks in the profiles were defined for their specific identification and differentiation. The two species could be readily differentiated based on their profile in the pSSU, the peak differences being associated with a nucleotide difference of