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Applied and Environmental Microbiology, January 2009, p. 108-112, Vol. 75, No. 1
0099-2240/09/$08.00+0     doi:10.1128/AEM.01341-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Terminal Restriction Fragment Length Polymorphism for Identification of Cryptosporidium Species in Human Feces {triangledown} ,{dagger}

L. S. Waldron,1* B. C. Ferrari,2,{ddagger} M. R. Gillings,1 and M. L. Power1

Department of Biological Sciences, Macquarie University, Sydney, New South Wales 2109, Australia,1 Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales 2109, Australia2

Received 16 June 2008/ Accepted 24 October 2008

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.

* Corresponding author. Mailing address: Biological Sciences, Macquarie University, Sydney NSW 2109, Australia. Phone: 612 9850 6974. Fax: 612 9850 8245. E-mail: lwaldron{at}bio.mq.edu.au

{triangledown} Published ahead of print on 31 October 2008.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

{ddagger} Present address: School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney NSW 2052, Australia.

Applied and Environmental Microbiology, January 2009, p. 108-112, Vol. 75, No. 1
0099-2240/09/$08.00+0     doi:10.1128/AEM.01341-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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