This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Google Scholar
Right arrow Articles by Brescia, C. C.
Right arrow Articles by Villegas, E. N.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Brescia, C. C.
Right arrow Articles by Villegas, E. N.
Agricola
Right arrow Articles by Brescia, C. C.
Right arrow Articles by Villegas, E. N.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, November 2009, p. 6856-6863, Vol. 75, No. 21
0099-2240/09/$08.00+0     doi:10.1128/AEM.00540-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Cryptosporidium Propidium Monoazide-PCR, a Molecular Biology-Based Technique for Genotyping of Viable Cryptosporidium Oocysts{triangledown}

Cristin C. Brescia,1 Shannon M. Griffin,2 Michael W. Ware,1 Eunice A. Varughese,1 Andrey I. Egorov,2 and Eric N. Villegas1*

National Exposure Research Laboratory,1 National Center for Environmental Assessment, U.S. Environmental Protection Agency, Cincinnati, Ohio 452682

Received 5 March 2009/ Accepted 28 August 2009

Cryptosporidium is an important waterborne protozoan parasite that can cause severe diarrhea and death in the immunocompromised. The current methods used to monitor for Cryptosporidium oocysts in water are the microscopy-based USEPA methods 1622 and 1623. These methods assess total levels of oocysts in source waters, but do not determine oocyst viability or genotype. Recently, propidium monoazide (PMA) has been used in conjunction with molecular diagnostic tools to identify species and assess the viability of bacteria. The goal of this study was the development of a Cryptosporidium PMA-PCR (CryptoPMA-PCR) assay that includes PMA treatment prior to PCR analysis in order to prevent the amplification of DNA from dead oocysts. The results demonstrated that PMA penetrates only dead oocysts and blocks amplification of their DNA. The CryptoPMA-PCR assay can also specifically detect live oocysts within a mixed population of live and dead oocysts. More importantly, live oocysts, not dead oocysts, were detected in raw waste or surface water samples spiked with Cryptosporidium oocysts. This proof-of-concept study is the first to demonstrate the use of PMA for pre-PCR treatment of Cryptosporidium oocysts. The CryptoPMA-PCR assay is an attractive approach to specifically detect and genotype viable Cryptosporidium oocysts in the water, which is critical for human health risk assessment.

* Corresponding author. Mailing address: Biohazard Assessment Research Branch, Microbiological and Chemical Exposure Assessment Research Division, National Exposure Research Laboratory, U.S. Environmental Protection Agency, Mailstop: 320, 26 W. Martin Luther King Dr., Cincinnati, OH 45268. Phone: (513) 569-7017. Fax: (513) 569-7117. E-mail: villegas.eric{at}epa.gov

{triangledown} Published ahead of print on 11 September 2009.

Applied and Environmental Microbiology, November 2009, p. 6856-6863, Vol. 75, No. 21
0099-2240/09/$08.00+0     doi:10.1128/AEM.00540-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»