Rapid identification of allergenic and pathogenic molds in environmental air by an oligonucleotide array

Abstract

Background: Airborne fungi play an important role in causing allergy and infections in susceptible people. Identification of these fungi, based on morphological characteristics, is time-consuming, expertise-demanding, and could be inaccurate.

Methods: We developed an oligonucleotide array that could accurately identify 21 important airborne fungi (13 genera) that may cause adverse health problems. The method consisted of PCR amplification of the internal transcribed spacer (ITS) regions, hybridization of the PCR products to a panel of oligonucleotide probes immobilized on a nylon membrane, and detection of the hybridization signals with alkaline phosphatase-conjugated antibodies.

Results: A collection of 72 target and 66 nontarget reference strains were analyzed by the array. Both the sensitivity and specificity of the array were 100%, and the detection limit was 10 pg of genomic DNA per assay. Furthermore, 70 fungal isolates recovered from air samples were identified by the array and the identification results were confirmed by sequencing of the ITS and D1/D2 domain of the large-subunit RNA gene. The sensitivity and specificity of the array for identification of the air isolates was 100% (26/26) and 97.7% (43/44), respectively.

Conclusions: Identification of airborne fungi by the array was cheap and accurate. The current array may contribute to decipher the relationship between airborne fungi and adverse health effect.

Figure 1

Layout of oligonucleotide probes on the array (0.8 × 0.7 cm, 9 by 8 dots). The probe "PC" was a positive control and the probe "NC" was a negative control (tracking dye only). The probe "M", a position marker, was an irrelevant probe labeled with a digoxigenin molecule at the 5' end. The corresponding species names and sequences of all probes are listed in Table 2. All probes used for fungal identification were spotted on the array in duplicate.

Figure 2

Hybridization patterns of 21 species of fungi on the array. The corresponding probes hybridized on the array are indicated in Figure 1, and the corresponding sequences of the hybridized probes are shown in Table 2. All probes used for fungal identification were spotted on the array in duplicate. The last two panels shows the simultaneous hybridization of two (Acremonium strictum and Stachybotrys chartarum) and three species (Scopulariopsis chartarum, Stachybotrys chartarum, and Trichoderma viride), respectively, on a single array.