Molecular Methods for Microsporidia Detection

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.


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. 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.