A Rapid and Specific Real-Time PCR Assay for the Detection of Clinically Relevant Mucorales Species

Abstract

Infections triggered by filamentous fungi placed in the order Mucorales, phylum Zygomycota, can cause serious harm to immunocompromised patients. Since there is lack of a standardized PCR (polymerase chain reaction) assay for early diagnosis of this fungal infection, this work was aimed to develop a new PCR assay able to detect the presence of Mucorales genera in clinical specimens. Here, we describe a novel diagnostic TaqMan MGB probe assay for precise and rapid detection of the most common clinical species of Mucorales. Zygomycete-specific oligonucleotides were designed to specifically amplify and bind highly conserved sequences of fungal 28S rRNA gene. Additionally, we succeeded in differentiating Mucorales species (i.e., Rhizopus, Lichtheimia, Mucor, and Rhizomucor) in artificially infected serum samples, suggesting that the quantitative capability of this real-time PCR assay could potentially optimize the diagnosis of mucormycosis.

Keywords: Mucorales; TaqMan MGB assay; diagnostic microbiology; filamentous fungi; mucormycosis; primer and probe design.

Figure 1

Amplification plot of Mucorales qPCR assays. The figure shows the triplicates of the analyzed samples. Green: Rhizopus; yellow: Mucor; blue: Lichtheimia; light blue: Rhizomucor; and light green: Cunninghamella.

Figure 2

Sensitivity of five different qPCR assays for detection of Mucorales. From left to right: Rhizopus; Mucor, Lichtheimia, Rhizomucor, and Cunninghamella.

Figure 3

Dynamic range of Mucorales quantification by qPCR. From left to right: Rhizopus, Mucor, Lichtheimia, Rhizomucor, and Cunninghamella.

Figure 4

Inhibitory test of Mucorales qPCR. The figure shows the amplification plot of the analyzed samples. Green: Rhizopus; yellow: Mucor; blue: Lichtheimia; light blue: Rhizomucor; light green: Cunninghamella; violet: p-IC.