Multiplex Single-Color PCR with Amplicon Melting Analysis for Identification of Aspergillus Species

Abstract

Multiplex, closed-tube detection of PCR products usually requires expensive probes of different colors. Alternatively, many PCR products can be distinguished by the amplicon melting temperature ( T m) in a single color with saturating DNA dyes. We describe here a T m-multiplexed assay for the identification and speciation of 4 Aspergillus species, A. fumigatus , A. flavus , A. niger , and A. terreus , plus an internal control that offers a promising method for rapid detection. Species identification is based on the amplicon melting patterns generated in the presence of the DNA saturating dye LCGreen® PLUS (Idaho Technology). Four Aspergillus species–specific forward primers were used with a consensus reverse primer. To separate all species, the forward primers for A. fumigatus and A. niger were tailed with additional nucleotides empirically chosen to increase the overall T m of the primers without contributing to nonspecific priming. Additionally, a plasmid of nonrelated sequence was used as an internal control, generating an amplicon with a higher T m than any of the Aspergillus species. Melting analysis was performed on either the LightCycler® (Roche) or the HR-1™ Instrument (Idaho Technology). We …