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

Abstract

BackgroundAirborne 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.MethodsWe 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.ResultsA 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.ConclusionsIdentification 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.

The Evaluation of Microbial Contamination in the Working Environment of Tanneries

“…The genetic analysis also indicated differences in the taxonomic classification of 1 bacteria species from genus Corynebacterium and even 5 mould species belonging to the genus of Penicillium selected as indicators of microbial contamination in the tanneries. The usefulness of molecular biology methods in the diagnostics of moulds isolated from the air has been proven (35,36).…”

Identification of Fungal Pathogens by Visible Microarray System in Combination with Isothermal Gene Amplification

“…Recently, microarray methods have been developed for the identification of a variety of pathogens, including viruses [ 17 ], bacteria [ 4 , 8 ], and fungi [ 5 – 7 ]. These methods are powerful tools to simultaneously detect multiple pathogens.…”

“…), pathogenic species of these genera have similar MIC values against several antifungal drugs, making designing genus-specific probes useful [ 18 – 21 ]. To our knowledge, the number of fungal species/genera that could be identified by our array system, 42 species from 24 genera, is the largest among microarray identification systems reported to date [ 5 – 7 ]. And the number of identifiable fungal species can be further increased depending on demand of clinical use.…”

Diagnostic Molecular Biology

“…The simplest arrays consist of spotting targets onto membranes using the Southern blotting platform developed years ago [49]. Numerous arrays of this type have been used to identify fungal species [50,51].…”