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Journal of Clinical Microbiology, November 2002, p. 3922-3928, Vol. 40, No. 11
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.11.3922-3928.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Real-Time PCR Method for Detection of Encephalitozoon intestinalis from Stool Specimens

D. M. Wolk,{dagger} S. K. Schneider, N. L. Wengenack, L. M. Sloan, and J. E. Rosenblatt*

Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota 55905

Received 22 March 2002/ Returned for modification 30 June 2002/ Accepted 12 August 2002

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 Encephalitozoon 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 Encephalitozoon intestinalis 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 102 to 104 spores/ml of feces, a value which represented a significant improvement over that achieved by staining (>=1.0 x 106 spores/ml). Melting temperature analysis of the amplicons allowed for the differentiation of three Encephalitozoon species (E. intestinalis, E. cuniculi, and E. hellem). The assay is readily adaptable to the clinical laboratory and represents the first real-time PCR assay designed to detect Encephalitozoon species.

* Corresponding author. Mailing address: Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic, 200 First St., SW, Rochester, MN 55905. Phone: (507) 284-2255. Fax: (507) 284-4272. E-mail: rosenblatt.jon{at}mayo.edu.

{dagger} Present address: Department of Pathology and Laboratory Medicine, Southern Arizona VA Health Care System, Tucson, AZ 85723.

Journal of Clinical Microbiology, November 2002, p. 3922-3928, Vol. 40, No. 11
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.11.3922-3928.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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