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

Abstract

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/μL), 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.

Molecular assays versus mycological methods for diagnosis of rhino orbital mucormycosis: analysis of 120 fresh clinical specimens from COVID-19 patients

“…The sensitivity and PPV of the specific SYBR Green-based PCR for the direct detection of mucormycosis from clinical samples were mostly concordant with the results of previous studies on the detection of Mucorales using multiplex real-time PCR in tissue specimens (31). As evidenced in our results, with the specific primers, the probability of misleading or unintended amplification concerning the usage of pan-fungal primers in tissue specimens particularly those with mixed infections or those collected from unsterile sites will be aborted (32). So, achieving a melting curve/amplification band in a molecular algorithm using Mucorales-specific primers in tissue biopsy could ascertain the diagnosis provided that both negative and positive yield the appropriate results (17).…”

A real‐time PCR for detection of pathogens of anthracnose on Chinese fir using TaqMan probe targeting ApMat gene

“…Four TaqMan probes were used in individual reactions to detect four different fungal pathogens, respectively. The TaqMan probes can be labeled with different reporter dyes, which allows multiplexing detection of two or more different pathogens in the same reaction 50–52 . In the future, the TaqMan probes, which hybridize to the ApMat target of C. cangyuanense , C. fructicola , C. gloeosporioides , and C. siamense , respectively, can be labeled with different reporter dyes and used in a multiplex reaction to detect four different pathogens of anthracnose on Chinese fir simultaneously to increase throughput.…”

Trans-kingdom fungal pathogens infecting both plants and humans, and the problem of azole fungicide resistance

“…Overall the use of qPCR based technics is highly recommended and encouraged for detecting Mucorales DNA, especially from serum (Rocchi et al, 2021 ). Mendonça et al ( 2022 ) developed a qPCR multiplex method for detecting R. arrhizus and four Aspergillus species. The qPCR is based on SYBR Green and a melting curve analysis.…”

“…The qPCR is based on SYBR Green and a melting curve analysis. It enables the simultaneous diagnosis of these invasive filamentous fungi either from culture or plasma, even when a small amount of DNA is available (Mendonça et al, 2022 ).…”