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Cryptosporidium Propidium Monoazide-PCR, a Molecular Biology-Based Technique for Genotyping Viable Cryptosporidium Oocysts
Citation:
Brescia, C., S. Hunt, M. W. WARE, E. VARUGHESE, A. EGOROV, AND E. VILLEGAS. Cryptosporidium Propidium Monoazide-PCR, a Molecular Biology-Based Technique for Genotyping Viable Cryptosporidium Oocysts . APPLIED AND ENVIRONMENTAL MICROBIOLOGY. American Society for Microbiology, Washington, DC, 75(21):6856-6863, (2009).
Impact/Purpose:
The overall objective of this task is the development of improved occurrence detection methods for protozoan parasites and Microsporidia. Since this work is a primary focus of the Branch, this task supports several individual projects related to sample preparation and protozoan detection. Together these projects will lead to complete methods able to support the UCMR and the CCL2 and CCL3.
Description:
Cryptosporidium is an important waterborne protozoan parasite that can cause severe diarrhea and death in the immunocompromised. Current methods to monitor for Cryptosporidium oocysts in water are 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 propidium monoazide-PCR (CryptoPMA-PCR) assay that includes PMA treatment prior to polymerase chain reaction (PCR) analysis in order to prevent the amplification of DNA from dead oocysts. The results demonstrated that PMA penetrates only heat-killed 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 and 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 in assessing human health risks associated with this pathogen.