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

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 (Tm) in a single color with saturating DNA dyes. We describe here a Tm-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 Tm 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 Tm than any of the Aspergillus species. Melting analysis was performed on either the LightCycler® (Roche) or the HR-1™ Instrument (Idaho Technology). We used Aspergillus primers that amplify 83- to 104-bp regions across the internal transcribed spacer 1 (ITS1) region and the 5.8S ribosomal RNA gene. The sequences are presented in Table 1 of the Data Supplement that accompanies the online version of this letter at http://www.clinchem.org/content/vol52/issue7, and alignments are presented in Fig. 1 of the online Data Supplement to show primer specificity. Representative derivative melting plots are presented in Fig. 11 . The use of tailed primers allowed good differentiation of the 4 Aspergillus amplicons. The mean Tm values for A. terreus, A. flavus, A. fumigatus, A. niger, and the internal control were well separated with SDs ≤0.35 (LightCycler) and ≤0.21 (HR-1). Reproducibility data are presented in Table 2 of the online Data Supplement. In this report, 2 of 4 sequence-specific forward primers were tailed to allow clear Tm-based differentiation of 4 Aspergillus species and an internal control with LCGreen PLUS. Differentiation of 2 related viral families based on multiplex melting curve analysis of amplicons with SYBR® Green I has been also been described (1). Furthermore, genotyping has been performed as a multiplex PCR with 3 primers, 2 of which were tailed with GC-rich sequences (2). However, in multiplex PCR, SYBR Green I preferentially detected high-Tm products and, compared with LCGreen, had poor resolution and broad melting transitions (3)(4). LCGreen has been used to differentiate 3 Mycobacterium species by amplicon Tm differences (5) and to genotype single-nucleotide variations in β-globin, cystic fibrosis, and other genes (4)(6). This Aspergillus assay is a single-color multiplexed assay with 7 primers and no probes. The use of LCGreen PLUS for the differentiation of amplicons with characteristic Tm is a simple and straightforward method for species identification. Aspergillus infections are common in immunocompromised patients and contribute significantly to mortality. Prompt treatment is essential, but rapid clinical diagnosis is difficult because symptoms are nonspecific and current laboratory methods are inadequate. A reliable method for early diagnosis provides a means for timely antifungal therapy. The combined use of PCR with antigen detection can aid in the diagnosis of invasive aspergillosis (7)(8). The simple method presented here provides rapid detection and speciation that can be applied to Aspergillus as well as other clinically relevant targets. Derivative melting plots obtained in a Tm-multiplexed PCR assay for Aspergillus speciation. Melting analyses were performed in the LightCycler (A) or the HR-1 instrument (B). The peaks in each plot correlate to A. terreus (a), A. flavus (b), A. fumigatus (c), A. niger (d), and an internal control (e). DNA was amplified in a LightCycler in multiplex format, with the primers given in Table 1 of the online Data Supplement. Tails were added to increase the Tms of A. fumigatus and A. niger. The PCR was performed on 2 μL of nucleic acid extracted from cultured mycelia and/or spores using the IDI Lysis Kit (GeneOhm Sciences) and further purified by use of the QIAamp DNA Blood Mini Kit (Qiagen). The DNA concentrations used in the PCR ranged from 4.6 to 10.4 ng/reaction. The total PCR volume was 10 μL and contained 20 mM Tris-HCl; 50 mM KCl; 0.25 mg/L bovine serum albumin; 2.0 mM MgCl2; 0.2 mM each of dATP, dTTP, and dGTP; 0.4 mM dUTP; 0.2 μM each of the forward Aspergillus primers, 0.2 μM reverse Aspergillus primer; 0.3 μM each of the internal control primers, 800 copies of internal control plasmid, 1× LCGreen PLUS; 0.5 U of UNG (Applied BioSystems), and 0.25 U of Platinum Taq (Invitrogen). Temperature cycling was performed with initial holds at 55 °C for 5 min and 95 °C for 1 min, followed by 60 cycles of 95 °C for 0 s, 64 °C for 0 s, and 72 °C for 3 s. After amplification, samples were heated to 95 °C for 5 s, cooled to 55 °C for 15 s, and slowly heated to 99 °C with continuous signal acquisition, followed by rapid cooling to 40 °C. Samples were stored at 4 °C until analyzed on the HR-1 Instrument with a temperature ramp rate of 0.3 °C/s and continuous fluorescence acquisition between 72 and 92 °C. This work was supported by the ARUP Institute for Clinical and Experimental Pathology. Aspects of high-resolution melting are licensed by the University of Utah to Idaho Technology and from Idaho Technology to Roche Applied Systems. C.T.W. holds equity interest in Idaho Technology.