Evaluation of Nucleic Acid Sequencing of the D1/D2 Region of the Large Subunit of the 28S rDNA and the Internal Transcribed Spacer Region Using SmartGene IDNS [Corrected] Software for Identification of Filamentous Fungi in a Clinical Laboratory

J Mol Diagn. 2012 Jul;14(4):393-401. doi: 10.1016/j.jmoldx.2012.02.004. Epub 2012 May 11.

Abstract

Filamentous fungal infections have recently increased because of the increasing numbers of immunocompromised hosts. In this study, we evaluated DNA sequencing of the D1/D2 region of the large subunit of the 28S ribosomal RNA gene and the internal transcribed spacer (ITS) region using SmartGene (SG; SmartGene Inc., Raleigh, NC) for the identification of a broad range of commonly encountered filamentous fungi. The SG proofreaders were used to upload, align, and edit fragments, and the resultant sequences were interpreted using the quality-controlled SG database. The results were compared with reference identifications using conventional phenotypic methods or ITS DNA sequences obtained from GenBank if phenotypic identifications were inconclusive. A total of 146 clinical isolates were included in this study, representing 49 different genera. The overall agreements of the D1/D2 and the ITS sequencing methods to reference identification were 97.2% (95% CI, 93.1% to 98.9%) and 97.7% (95% CI, 92.8% to 99.4%), respectively. Of the 146 isolates, 18 (12.3%) did not amplify using the ITS universal primers after repeated attempts and, therefore, could not be sequenced using this target. Correct identification was achieved for 100% (95% CI, 97.4% to 100%) of the isolates when applying both the D1/D2 and ITS targets. In summary, DNA sequencing using SG software provides a rapid, accurate, and reliable tool for the identification of filamentous fungi in a clinical laboratory.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA, Fungal / genetics
  • DNA, Ribosomal / genetics*
  • DNA, Ribosomal Spacer / genetics*
  • Fungi / genetics*
  • RNA, Ribosomal, 28S / genetics*
  • Sequence Analysis, DNA / methods*
  • Software*

Substances

  • DNA, Fungal
  • DNA, Ribosomal
  • DNA, Ribosomal Spacer
  • RNA, Ribosomal, 28S