Development of PCR real-time MGB method for detection of zygomycete fungi

Objectives. “Zygomycosis” represents a wide spectrum of clinical disease types, which refers to infections caused by fungi classifiable as zygomycetes. It has been estimated that the incidence of zygomycosis is relatively low, but it constitute a significant area of concern, especially in immunocompromised patients. One of the major problems concerning zygomycosis is the early accurate diagnosis and the identification of the organism involved. Since no routine serologic tests for the diagnosis of zygomycosis are currently available and DNAbased techniques for detection are not yet fully standardized or commercially available the aim of this study was to develop new procedures to improve the prognosis of these infections. Methods. A new quantitative PCR-MGB (qPCR) real-time assay system was developed to detect Rhizopus spp., Mucor spp., Absidia spp., and Rhizomucor spp. The tested strains were cultured, and DNA was extracted and amplified. Primers and TAQMAN MGB probes were designed to detect a conserved region of the 28S ribosomal RNA sequence zygomycetes. Positive control plasmids was obtained by Twin Helix (Rho, Italy) and named pZIGO1 (Rhizopus, Absidia, Mucor), pZIGO2 (Rhizomucor), pIC (Jellyfish sequence). Real-time PCR amplification was performed by a 7500 Real Time PCR System (Applied Biosystems). The sensitivity was defined by the lowest concentration of target quantified at a frequency of 100%.The dynamic range was evaluated using 10-fold dilutions (from 10^9-10^1 copies/reaction) of pZIGO1 and pZIGO2 plasmids. Results. We developed quantitative real-time MGB-PCRs that targeted the most common zygomycetes causing human disease. The RTPCR assay was able to quantify Rhizopus from 10^9 to 10^1 copies/reaction with a dynamic range of 10^9-10^1 copies/reaction (R2 = 0.999); Mucor from 10^9 to 10^1 copies/reaction with a dynamic range of 10^9-10^1 copies/reaction (R2 = 0.999); Absidia from 10^9 to 10^2 copies/reaction with a dynamic range of 10^9-10^1 copies/reaction (R2 = 0.990) and Rhizomucor from 10^9 to 10^2 copies/reaction with a dynamic range of 10^9-10^1 copies/reaction (R2 = 0.990). Additionally, we were able to detect the zygomycetes DNA in serum samples obtained by spike in with a known amount of Rhizopus, Absidia, Mucor, and Rhizomucor conidia. Conclusion. In situ hybridization, conventional PCR and nucleic acid sequencing suffer from a relatively long turnaround time compared with that required for the progression of zygomycetes infections. The application of real-time PCR methodologies is attractive due to increase in sensitivity, reduction of outside contamination, and decreased test turnaround time. We have developed a real-time PCR TAQMAN that is highly specific for clinically significant zygomycetes genera. Future studies are currently being designed to validate the potential value of these qPCR assays in a clinical setting to improve the diagnostic specificity and therapeutic monitoring of disease progression or resolution.