Molecular Methods for Microsporidia Detection Use of an Inhibitor Control With Real-Time PCR - Water Research Foundation Report Series

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.