Terminal Restriction Fragment Length Polymorphism for Identification of Cryptosporidium Species in Human Feces

Effective management of human cryptosporidiosis requires efficient methods for detection and identification of the species of Cryptosporidium isolates. Identification of isolates to the species level is not routine for diagnostic assessment of cryptosporidiosis, which leads to uncertainty about the...

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Bibliographic Details
Published in:Applied and Environmental Microbiology 2009-01, Vol.75 (1), p.108-112
Main Authors: Waldron, L.S, Ferrari, B.C, Gillings, M.R, Power, M.L
Format: Article
Language:English
Subjects:
Animals
Biological and medical sciences
Cryptosporidiosis
Cryptosporidiosis - parasitology
Cryptosporidium - classification
Cryptosporidium - genetics
Cryptosporidium - isolation & purification
Cryptosporidium parvum
DNA Fingerprinting - methods
DNA, Protozoan - genetics
DNA, Ribosomal - genetics
Feces
Feces - parasitology
Fundamental and applied biological sciences. Psychology
Gastroenterology
Genes
Genotype
Humans
Methods
Microbiology
Parasitic protozoa
Polymorphism
Polymorphism, Genetic
Polymorphism, Restriction Fragment Length
Ribonucleic acid
RNA
RNA, Ribosomal, 18S - genetics
Sensitivity and Specificity
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Summary:Effective management of human cryptosporidiosis requires efficient methods for detection and identification of the species of Cryptosporidium isolates. Identification of isolates to the species level is not routine for diagnostic assessment of cryptosporidiosis, which leads to uncertainty about the epidemiology of the Cryptosporidium species that cause human disease. We developed a rapid and reliable method for species identification of Cryptosporidium oocysts from human fecal samples using terminal restriction fragment polymorphism (T-RFLP) analysis of the 18S rRNA gene. This method generated diagnostic fragments unique to the species of interest. A panel of previously identified isolates of species was blind tested to validate the method, which determined the correct species identity in every case. The T-RFLP profiles obtained for samples spiked with known amounts of Cryptosporidium hominis and Cryptosporidium parvum oocysts generated the two expected diagnostic peaks. The detection limit for an individual species was 1% of the total DNA. This is the first application of T-RFLP to protozoa, and the method which we developed is a rapid, repeatable, and cost-effective method for species identification.
ISSN:0099-2240
1098-5336
1098-6596
DOI:10.1128/AEM.01341-08