Real-Time PCR Method for Detection of Zygomycetes

• Published in: Journal of Clinical Microbiology Vol. 46; no. 7; pp. 2353 - 2358
• Main Authors: Hata, D. Jane, Buckwalter, Seanne P, Pritt, Bobbi S, Roberts, Glenn D, Wengenack, Nancy L
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.07.2008; American Society for Microbiology (ASM)
• Subjects: Absidia; Aspergillus; Biological and medical sciences; Candida; Cunninghamella; Cytochromes b - genetics; DNA Primers - genetics; Fluorescence Resonance Energy Transfer; Fundamental and applied biological sciences. Psychology; Fungal Proteins - genetics; Fungi - genetics; Fungi - isolation & purification; Fusarium; Humans; Microbiology; Mucor; Mucor racemosus; Mycology; Mycoses - diagnosis; Mycoses - microbiology; Nucleic Acid Hybridization - methods; Polymerase Chain Reaction - methods; Polymerase Chain Reaction - standards; Reference Standards; Rhizopus; Sensitivity and Specificity; Transition Temperature; Zygomycetes; Polymerase chain reaction; Method; Microbiology; Detection; Real time
• ISSN: 0095-1137; 1098-660X; 1098-5530
• DOI: 10.1128/jcm.02331-07

Zygomycete infections can be devastating in immunocompromised hosts. Difficulties in the histopathologic differentiation of this class from other filamentous fungi (e.g., Aspergillus spp., Fusarium spp.) may lead to delays in diagnosis and initiation of appropriate treatment, thereby significantly affecting patient outcome. A real-time PCR assay was developed to detect species of the zygomycete genera Absidia, Apophysomyces, Cunninghamella, Mucor, Rhizopus, and Saksenaea in culture and tissue samples. Primers and fluorescence resonance energy transfer hybridization probes were designed to detect a 167-bp conserved region of the multicopy zygomycete cytochrome b gene. A plasmid containing target sequence from Mucor racemosus was constructed as a positive control. The analytical sensitivity of the assay is 10 targets/μl, and a specificity panel consisting of other filamentous fungi, yeasts (Candida spp.), and bacteria demonstrated no cross-reactivity in the assay. The clinical sensitivity and specificity of the assay from culture isolates were 100% (39/39) and 92% (59/64), respectively. Sensitivity and specificity determined using a limited number of fresh tissue specimens were both 100% (2/2). The sensitivity seen with formalin-fixed, paraffin-embedded tissues was 56% (35/62), and the specificity was 100% (19/19). The speed, sensitivity, and specificity of the PCR assay indicate that it is useful for the rapid and accurate detection of zygomycetes.