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Microbiological and Chemical Exposure Assessment

Research In Action

Evaluation of a novel approach using propidium monoazide for detecting viable microorganisms

Issue

Molecular methods that detect nucleic acids are increasingly being used to identify microbial contaminants in the environment. While these new techniques are more rapid and sensitive than traditional approaches, they cannot be used to determine if the detected organism is active and infectious. Without this information, developing accurate risk assessments is difficult.

Action

EPA scientists are conducting several projects designed to test the efficacy of propidium monoazide (PMA)  a nucleic acid intercalating dye that does not penetrate the membranes of live cells or the capsids of intact viruses. Specifically, scientists are determining if PMA can prevent amplification of nucleic acids from a range of non-viable, environmentally relevant microorganisms by polymerase chain reaction (PCR).

These studies have tested a wide range of microorganisms, including bacteria, fungi, protozoa, and viruses, to determine if PMA is effective with different types of organisms. This technique may also lead to the development of more rapid environmental occurrence methods. Current viability assays require the use of animal bioassay or cell culture, both of which take days to weeks to complete, while the PMA treatment adds less than an hour to a molecular method. Such a method would be valuable to the environmental research community by allowing for more regular use of molecular detection methods in occurrence studies.

Result and Impact

PMA has been used to specifically detect viable (e.g., intact or undamaged) organisms including norovirus, enterovirus, fecal indicator bacteria, Cryptosporidium, Giardia and fungal species. Additional work is being conducted to incorporate the PMA treatment technique into several other microbial occurrence methods.

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