Application of a multiplex suspension array for rapid and simultaneous identification of clinically important mold pathogens

We have developed a microsphere-based suspension array (MSA) for the identification of 23 medically important mold pathogens including Aspergillus spp., Fusarium spp., Mucor spp., Rhizopus spp., Rhizomucor pusillus, Penicillium marneffei, Saksenaea vasiformis, Apophysomyces elegans, Lichtheimia corymbifer, and Syncephalastrum racemosum. Twenty-one oligonucleotide probes were designed based on the internal transcribed spacer (ITS2) region for species level identification of molds. Among the 21 probes, 2 probes are shared by more than one species due to low or absence of sequence variability, i.e. Rpam for Rhizopus azygosporus/Rhizopus microsporus and Fumop for Fusarium moniliforme/Fusarium oxysporum/Fusarium pallidoroseum. No cross reactivity was identified except for probes of Mucor racemosus (Murac) which cross react with Mucor hiemalis and Mucor ramosissimus. The sensitivity of MSA is 100 fg–1 ng. The whole procedure including DNA extraction and PCR amplification can be finished within 5 h. The MSA is simple, rapid, specific, high-throughput and capable of multiple-species detection in one reaction tube.