Andreas Nocker, Mark Burr, and Anne Camper

OBJECTIVES:

The purpose of this project was to review the literature on the potential for molecular methods to be adopted by the drinking water industry for monitoring microbiological water quality.

BACKGROUND:

The implementation of molecular techniques is growing rapidly in clinical and environmental microbiology. Some methods detect pathogenic organisms (culturable or not) within a few hours, instead of the days required by cultivation. It would be ideal to monitor all pathogens starting with one single sample. Pathogen detection and quantification methods should be rapid, sensitive, highly accurate, easy to perform, and amenable to high-throughput analyses. This document discusses the advantages and disadvantages of current and emerging molecular methods relevant to the water industry and their potential for replacing or supplementing traditional cultural methods.

HIGHLIGHTS:

Molecular methods have not been adopted because of regulatory inertia, satisfaction with the current system, lack of standardization, relatively high costs, and lack of trained personnel. Molecular methods are prone to inhibition, do not generally distinguish live from dead cells, and may produce ambiguous results. Molecular technology should be automated for continuous online monitoring. Biosensor research has not targeted the water industry and has not adequately addressed sample concentration and processing. The experts surveyed for this project recommended more fundamental research into molecular methods using real samples, efforts toward standardization, and promoting dialogue among utility personnel, regulators, drinking water experts, and molecular biologists. The experts believe five to ten years of development and testing would be required before molecular methods would be adopted for routine water analysis.

APPROACH:

The work was accomplished through literature searches, contacting companies producing molecular detection methods, and a survey of experts and utilities. Specific molecular methods were summarized and the advantages and disadvantages reported.

RESULTS/FINDINGS:

Currently, no molecular technology exists that can be directly transferred to the water industry. Many problems still exist but components are present that could be incorporated into the "ideal" system. This should be an automated system capable of sampling large volumes of water that integrates sample concentration, processing, and detection in a single platform. Immunomagnetic separation (IMS) is recommended because it permits removal of contaminants and adds specificity. Immuno-captured organisms should be detected via nucleic acids and greater research into purification of nucleic acids (especially RNA) from water samples is needed.

Although molecular methods are often perceived as highly sensitive, poor sensitivity is actually an obstacle to implementation. To increase sensitivity, nucleic acid amplification will probably be necessary. Nucleic acid sequence-based amplification (NASBA) rather than PCR is best because NASBA detects RNA (a test for cell viability), may be less prone to inhibition, and operates isothermally (more compatible with automation). Quantum dots (QDs) have the potential for increasing the sensitivity of molecular assays and provide the ultimate in multiplexing capability. Regulators, researchers, and utilities should discuss how the results of molecular assays will be interpreted and affect water treatment and public health decisions. Companies that develop molecular technology have advanced the field, but there is a tendency to oversell technology. Research groups with promising technology should be encouraged to perform collaborative validation with utilities.

IMPACT:

This project summarizes the relevant molecular methods that are available to the drinking water industry and summarizes their advantages and disadvantages. It also illustrates where more work is needed (removal of inhibitors, increasing specificity, increasing acceptance, etc.). These findings also show that molecular methods are not yet ready for use by most utilities, and are not currently at a state where they can be used for regulatory compliance. There are many reasons why molecular methods would be a good tool for regulatory compliance of microbiological water quality, but more research and testing will be required.

RESEARCH PARTNER:

U.S. Environmental Protection Agency