Optimization of a Quantitative PCR Methodology for Detection of Aspergillus spp. and Rhizopus arrhizus

Introduction Multiplex quantitative polymerase chain reaction (qPCR) methods for the detection of Aspergillus spp. based only on SYBR Green and melting curve analysis of PCR products are difficult to develop because most targets are located within ITS regions. The aim of this study was to adapt our previously developed methodology based on a multiplex PCR assay coupled with GeneScan analysis to provide a qPCR method. Methods A SYBR Green-based real-time PCR assay was optimized to detect A. fumigatus, A. flavus, A. niger, A. terreus, and R. arrhizus in a multiplex assay and applied to cultured fungi and spiked plasma. Results Different melting temperatures allowed identification of all five pathogens and discrimination between them, even in samples with low amounts of fungal gDNA (from 1.3 to 33.0 pg/mu L), which has been reported previously as problematic. No false-positive results were obtained for non-target species, including bacteria and human DNA. This method allowed detection of fungal pathogens in human plasma spiked with fungal DNA and in coinfections of A. niger/R. arrhizus. Discussion This work provides evidence for the use of a qPCR multiplex method based on SYBR Green and melting curve analysis of PCR products for the detection of A. fumigatus, A. flavus, A. niger, A. terreus, and R. arrhizus. The proposed method is simpler and less expensive than available kits based on fluorescent probes and can be used for aiding diagnosis of the most relevant invasive filamentous fungi, particularly in low-income health care institutions.

Automated summary

This study developed a qPCR method for the detection of multiple pathogens using SYBR Green and melting curve analysis. The method was able to accurately identify different fungal pathogens even in samples with low amounts of fungal gDNA. It did not produce false-positive results for non-target species and can be a simpler and more cost-effective alternative for diagnosing invasive filamentous fungi, especially in low-income healthcare institutions.