Ordering Information:
ORDER NUMBER:  91185
DATE AVAILABLE: Spring 2008

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PRINCIPAL INVESTIGATORS:
Donna M. Wolk, Greg Sturbaum, Rebecca Hoffman, Charles R. Sterling, and Marilyn M. Marshall

OBJECTIVES:
The objective of this project was to optimize a robotic nucleic acid extraction method and design a real-time polymerase chain reaction (PCR) assay to detect microsporidian spores, which are currently listed on the U.S. Environmental Protection Agency’s Contaminant Candidate List (CCL). To accomplish this objective, the project team compared the efficiency of the nucleic acid extraction methods with those previously used for extraction of microsporidian spores in laboratory water and feces.

BACKGROUND:
Detection of microorganisms in the U.S. water supply is crucial to the prevention of waterborne disease and bioterrorism. There is a current and critical need for optimized and standardized methods and evidence-based comparisons of methods to detect waterborne pathogens. Despite the advantages that molecular methods offer for improved sensitivity, specificity, and species confirmation, historical microscopic methods are considered to be the gold standard; molecular detection of microbes from source water is an ongoing area of research and no consensus methods currently exist. If molecular methods, such as polymerase chain reactions (PCR), are to supplement and/or replace microscopic methods, new methods must be verified and validated according to the highest standards of both sensitivity and specificity, so that false-negative and false-positive results may be avoided and evidence based risk-management decisions can be made. 

HIGHLIGHTS:
A real-time PCR method, labeled Encephalitozoon-PCR (ENC-PCR) and designed for detection of microsporidian spores (i.e., Encephalitozoon sp.), successfully amplified ribosomal DNA (rDNA) from Ecephalitozoon intestinalis spores spiked into laboratory and source water (10L grab sample pellets). The analytical sensitivity was as low as 5–10 spores/sample in 10L grab samples of Colorado source water. The method was successfully optimized to include an exogenous internal control target, an Inhibitor Control (IC) to identify the presence of PCR inhibitors in sample matrices. The ENC-PCR represents the first statistically characterized real-time assay designed to detect microsporidia in source water samples while identifying the presence of PCR inhibitors.

APPROACH:
Using laboratory water spiked with E. intestinalis spores, robotic nucleic acid extraction methods were compared by using numeric cycle threshold output values (Ct values), which are indirectly proportional to the target DNA concentration in a real-time PCR. The Ct values were used to guide decisions for optimizing the method’s performance under various experimental conditions. The method’s analytical sensitivity was determined in laboratory water. An additional synthetic oligonucleotide target was then added to serve as an IC. IC Cycle threshold values (IC-Ct) were used to monitor source water samples for the presence of PCR inhibitors, which could cause false negative results. Standardized protocols were documented and the PCR method was used to detect microsporidian spores spiked into various source water samples.

RESULTS/FINDINGS:
The ENC-PCR assay’s lower confidence limit in source water (the spore concentration that yields positive reactions in ≥ 90 percent of cases) was as low as 5–10 spores/sample in Colorado source water and a lower detection limit as low as 1 spore/sample in non-inhibitory water matrices. ENC-PCR efficiency of 85–95 percent was achieved in laboratory water; 51–90 percent in source water. The ENC-PCR assay was successfully optimized to include an exogenous internal control target as an IC to identify the presence of PCR inhibitors in sample matrices. The performance of the IC was successful in identification of PCR inhibitors in source water matrices. When PCR inhibitors were detected, addition of 2X bovine serum albumin to the PCR master mix improved PCR efficiency in source water.

IMPACT:
The ENC-PCR represents the first statistically characterized real-time assay designed to detect microsporidia in source water samples while identifying the presence of PCR inhibitors. The ability to identify PCR inhibitors may provide the water industry with a measure of confidence, previously undefined for PCR, providing for assessment of false negative PCR reactions and enhancing risk assessment capabilities. The ENC-PCR is a useful tool for detection of microsporidia in source water and may be adaptable for standardization and use by the USEPA. Future efforts should be aimed at validating the ENC-PCR assay performance in different laboratories.

PARTICIPANTS:
Denver Water and the City of Fort Collins participated in this project.