Real-Time PCR Method for Detection of

Abstract:

The prevalence of microsporidiosis is likely underestimated due to the labor-intensive, insensitive, and nonspecific clinical laboratory methods used for the diagnosis of this disease. A real-time PCR assay was designed to assess DNA extraction methods and to detect three species in feces. Modifications of the MagNA Pure LC DNA isolation kit protocol (Roche Applied Sciences, Indianapolis, Ind.) were compared by using the automated MagNA Pure LC instrument (Roche) and fecal specimens spiked with spores. Extracted DNA was amplified by the LightCycler (Roche) PCR assay. Assay sensitivity, reproducibility, and efficiency were assessed by comparing threshold crossover values achieved with different extraction and storage conditions (fresh, refrigerated, frozen, and preserved specimens). Optimal extraction conditions were achieved by using a commercial buffer, tissue lysis buffer (Roche), as the specimen diluent. LightCycler PCR results were compared to those obtained from routine stool microscopy with trichrome blue stain. The lower limit of detection for the LightCycler PCR assay varied by storage conditions from 10 to 10 spores/ml of feces, a value which represented a significant improvement over that achieved by staining (�.0 × 10 spores/ml). Melting temperature analysis of the amplicons allowed for the differentiation of three species (. , . , and . ). The assay is readily adaptable to the clinical laboratory and represents the first real-time PCR assay designed to detect species.

Journal of Clinical Microbiology.2002:40(11):3922

Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Roc

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