Full Length Research Paper

Evaluation and guidelines for use of polymerase chain reaction-computer database homology comparison (PCR-CDHC) for detection and species determination of human pathogenic microsporidia

Scot E. Dowd¹* and Jeanette A. Thurston-Enriquez<sup>2

1</sup>USDA-ARS, Livestock Issues Research Unit, Lubbock, TX, Texas.
²USDA-ARS, Soil and Water Conservation Research Unit, Lincoln, NE 68583, Texas.

*Corresponding author. E-mail: sdowd@lbk.ars.usda.gov. Tel: 806-746-5356 ext 122 office. Fax: 806-744-5028.

Accepted 11 January, 2008
 

The potential for waterborne disease and zoonotic transmission of at least two species of human pathogenic microsporidia has heightened interest in clinical and environmental detection methods for these organisms. Detection using the polymerase chain reaction (PCR) followed by computer database homology comparison (CDHC) (PCR-CDHC) was reported previously by this research group. As a result, PCR-CDHC has been employed by many research groups around the world for species determination of human pathogenic microsporidia. To validate the CDHC speciation approach, a phylogenetic tree was generated using the small subunit ribosomal DNA sequences (SSU-rDNA) of a large number of microsporidia. An index of similarity was created and used as part of an assessment of CDHCs ability to differentiate between closely related species. Polymerase chain reaction followed by dye termination PCR sequencing and subsequent CDHC of the sequences was performed on 8 species of microsporidia including four human pathogenic strains. The four non-human pathogenic microsporidia tested by this approach were those shown by the phylogenetic analyses to be very closely related to the other human pathogenic species as determined by branch length. In all cases the CDHC approach was able to correctly identify the eight species of microsporidia evaluated. To provide an example of PCR-CDHC, a “universal” and two previously published pathogen-specific microsporidia PCR protocols followed by PCR-CDHC was conducted to assess their ability to detect naturally occurring microsporidia species in swine wastewater. Only one primer set resulted in a PCR-CDHC analysis where presumptive human pathogenic microsporidia was detected. Subsequent CDHC showed these presumptive positive PCR results were actually false positives. With the appropriate primer set, PCR-CDHC proves to be a reliable method that can be used for specific species determination of human pathogenic microsporidia in samples where non-pathogenic species may be present.

Key words:  Microsporidia, Sequencing, BLAST, PCR, detection.