Terminal restriction fragment length polymorphism for identification of cryptosporidium species in human feces

L.S. Waldron, B. C. Ferrari, M. R. Gillings, M. L. Power

Research output: Contribution to journalArticleResearchpeer-review

Abstract

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.

LanguageEnglish
Pages108-112
Number of pages5
JournalApplied and Environmental Microbiology
Volume75
Issue number1
DOIs
Publication statusPublished - Jan 2009

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Cryptosporidium
Feces
feces
Restriction Fragment Length Polymorphisms
restriction fragment length polymorphism
polymorphism
Cryptosporidiosis
cryptosporidiosis
Oocysts
genetic polymorphism
oocysts
Cryptosporidium hominis
methodology
Cryptosporidium parvum
rRNA Genes
human diseases
Uncertainty
Protozoa
Limit of Detection
epidemiology

Cite this

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abstract = "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.",
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Terminal restriction fragment length polymorphism for identification of cryptosporidium species in human feces. / Waldron, L.S.; Ferrari, B. C.; Gillings, M. R.; Power, M. L.

In: Applied and Environmental Microbiology, Vol. 75, No. 1, 01.2009, p. 108-112.

Research output: Contribution to journalArticleResearchpeer-review

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