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Use of LightCyclerTM PCR to evaluate two stool extraction methods for detection of microsporidia in fresh and preserved specimens



Use of LightCyclerTM PCR to evaluate two stool extraction methods for detection of microsporidia in fresh and preserved specimens



Abstracts of the General Meeting of the American Society for Microbiology 101: 223-224



The prevalence of microsporidian disease is likely underestimated due to labor-intensive, insensitive and non-specific laboratory methods used for diagnosis. A LightCyclerO real-time PCR (LC-PCR) assay was designed to detect and confirm the identity of microsporidia in feces and other body fluids. Using fecal specimens, a modification of the automated Roche MagNA Pure LC DNA Isolation Kit I protocol was compared to QIAGEN QIAamp(R) DNA Stool Mini-Kit method. Clinical and analytical sensitivity of the assay was determined by performing serial dilutions of Encephalitozoon spores in fresh, frozen, and preserved fecal matrixes and in water; extracting DNA; and performing LC-PCR. Reproducibility of extraction methods and PCR was determined by monitoring the threshold (cross-over) cycle number of the LC-PCR. Both methods successfully extracted microsporidian DNA from specimens for amplification; the median limit of detection (LOD) was 1.0+E4 spores/ml in fresh and frozen feces (n=8). In contrast, the Ryan Chromotrope 2R stain LOD was gtoreq1.0+E6 spores/ml (n=3). Both extraction methods had consistent extraction efficiencies and were effective for removal of most PCR inhibitors. Both extraction methods, combined with LC-PCR, detected E. cuniculi, E. hellem, and two strains of E. intestinalis and provided species identification based on reproducible differences in the amplicon melting temperatures. DNA amplification was also possible using stool specimens spiked with microsporidia (1.0+E7 spores/ml feces) and stored in EcoFixTM preservative for 72 hrs (n=3).

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