Development of multiplex real-time PCR for rapid identification and quantitative analysis of Aspergillus species

Abstract

The identification of Aspergillus species and azole resistance is highly important for the treatment of invasive aspergillosis (IA), which requires improvements in current fungal diagnostic methods. We aimed to develop multiplex real-time PCR to identify major Aspergillus section and azole resistance. BenA and cyp51A genes were used to design primers, probes, and control DNA for multiplex PCR. Qualitative and quantitative analysis was conducted for 71 Aspergillus and 47 non-Aspergillus isolates. Further, the limit of detection (LOD) and limit of quantitation (LOQ) from hyphae or conidia were determined according to the culture time. Newly developed real-time PCR showed 100% specificity to each Aspergillus section (Fumigati, Nigri, Flavi, and Terrei), without cross-reaction between different sections. In quantitative analysis of sensitivity measurements, LOD and LOQ were 40 fg and 400 fg, respectively. Melting temperature analysis of the cyp51A promoter to identify azole resistance showed temperatures of 83.0 ± 0.3˚C and 85.6 ± 0.6˚C for susceptible A. fumigatus and resistant isolates with TR34 mutation, respectively. The minimum culture time and fungal colony size required for successful detection were 24 h and 0.4 cm in diameter, respectively. The developed multiplex real-time PCR can identify common Aspergillus sections quantitatively and detect presence of the TR34 mutation. Further, this method shows high sensitivity and specificity, allowing successful detection of early-stage fungal colonies within a day of incubation. These results can provide a template for rapid and accurate diagnosis of IA.

The Aspergillus niger Major Allergen (Asp n 3) DNA-Specific Sequence Is a Reliable Marker to Identify Early Fungal Contamination and Postharvest Damage in Mangifera indica Fruit

“…The pathogenicity assay results obtained here showed that the mango fruit is very susceptible to contamination with A. niger , and the type of injuries caused by fungal colonization is compatible with that described by other authors ( Kim et al, 2020 ; Shen et al, 2020 ). These results confirm the validity and applicability of this method for the study of the spoilage of mangoes by A. niger .…”

Morphological and genetic identification of fungal genus/species in formalin‐fixed, paraffin‐embedded specimens obtained from patients with histologically proven fungal infection

“…Real-time PCR targeting the fungal genome has also been used to identify fungal species in FFPE samples in several studies. [22][23][24][33][34][35] Some of these studies employed a multiple detection system for several fungi in FFPE samples. [22][23][24] A multiplex real-time PCR system targeting several fungal genomes showed an overall sensitivity of 64% for the identification of fungi from FFPE specimens.…”

“… 22‐24,33‐35 Some of these studies employed a multiple detection system for several fungi in FFPE samples. 22‐24 A multiplex real‐time PCR system targeting several fungal genomes showed an overall sensitivity of 64% for the identification of fungi from FFPE specimens. 24 The authors focused on filamentous fungi and succeeded in identifying Aspergillus spp.…”

“…18,19 However, because nucleic acid is highly fragmented in FFPE samples, sensitivity of PCR in FFPE samples was not high for determining fungal genus/ species. 20,21 Multiple real-time PCR targeting short fragments of fungal genomes has recently been used to simultaneously identify several fungi in FFPE samples [22][23][24] ; however, the number of fungal genus/species is limited with this technique. Detailed morphological features specific to fungal genus/species in FFPE samples have not been well described because fungal genus/species cannot be easily identified in FFPE samples.…”

Multiplex PCR Based Strategy for Detection of Fungal Pathogen DNA in Patients with Suspected Invasive Fungal Infections

“…Results showed that the proposed method detected correctly all ten targeted pathogens, exhibited no cross-reactivity with nontargeted species and presented a limit of detection of 10 to 1 pg of DNA. The limit of detection in PCR assays is highly dependent on the method used to detect the amplified fragments, being much lower for pPCR and GeneScan analysis, in the range of pg to fg of DNA, compared with conventional electrophoresis (agarose or polyacrylamide gels), usually ng of DNA [ 39 ]. This multiplex panel for filamentous fungi was able to distinguish Aspergillus species and R. arrhizus, which has been a problem in other multiplex strategies [ 40 ].…”