Identification of medically important molds by an oligonucleotide array

Abstract

Infections caused by fungi have increased in recent years. Accurate and rapid identification of fungal pathogens is important for appropriate treatment with antifungal agents. On the basis of the internal transcribed spacer 1 (ITS 1) and ITS 2 sequences of the rRNA genes, an oligonucleotide array was developed to identify 64 species (32 genera) of clinically important filamentous (or dimorphic) fungi. These 64 species included fungi causing superficial, cutaneous, subcutaneous, and invasive infections. The method consisted of PCR amplification of the ITS regions using a pair of universal primers, followed by hybridization of the digoxigenin-labeled PCR products to a panel of species- or group-specific oligonucleotides immobilized on a nylon membrane. Of 397 fungal strains (290 target and 107 nontarget strains) tested, the sensitivity and specificity of the array was 98.3% (285/290) and 98.1% (105/107), respectively. Misidentified strains were usually those belonging to the same genus of the target species or having partial homology with oligonucleotide probes on the membrane. The whole procedure can be finished within 24 h starting from isolated colonies; reproductive structures, which are essential for the conventional identification methods, are not needed. In conclusion, the present array is a powerful tool for identification of clinically important filamentous fungi and may have the potential to be continually extended by adding further oligonucleotides to the array without significantly increasing the cost or complexity.

FIG. 1.

Layout of oligonucleotide probes on the array (1.1 by 0.9 cm). The probe “PC” (G10) was designed on the basis of the ITS 2 region of Saccharomycodes ludwigii BCRC 21378 and used as a positive control. Probes coded “NC” (E6 and G2 to G4) were negative controls (tracking dye only). Probes coded “M” were ITS 4 labeled with digoxigenin at its 5′ end and were used as position markers. Group-specific probes are underlined, and multiple probes used to identify a single species are in bold face. The corresponding sequences of probes are listed in Table 1.