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Using Propidium Monoazide as an Enrichment Step to Remove Extraneous DMA and Non-Viable Organisms for Cryptosporidium Genotyping
Citation:
VILLEGAS, E. Using Propidium Monoazide as an Enrichment Step to Remove Extraneous DMA and Non-Viable Organisms for Cryptosporidium Genotyping. Presented at Global Conference on Waterborne Microbial Contaminants, Singapore, SINGAPORE, October 05 - 08, 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 protozoan parasite that continues to cause waterborne disease outbreaks worldwide. 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-polymerase chain reaction (CryptoPMA-PCR) assay that includes PMA treatment prior to PCR analysis. The purpose of the treatment was to prevent the amplification of DNA that was extraneous or in inactivated oocysts. Our results demonstrated that PMA effectively removes naked DNA present in environmental samples. In addition, this CryptoPMA-PCR assay can specifically detect live oocysts within a mixed population of live and dead oocysts. More importantly, only live oocysts were detected in raw waste or surface water samples that were spiked with either live or dead 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.