Development and Validation of a Real-Time PCR Assay for the Detection of Aspergillus fumigatus on Panther Fusion System

Abstract

This protocol aims to provide a detailed description of the development and clinical validation of a laboratory-developed test (LDT) for the detection of Aspergillus fumigatus in respiratory specimens using the Panther Fusion Open Access system. This platform allows automated extraction of the pathogen genomic DNA and amplification of targets. Specifically, the assay targets the ITS1 region of fungal ribosomal DNA through a real-time PCR workflow. Specificity of primers and probe was verified in silico, and assay parameters, such as MgCl₂, KCl, primer/probe concentrations, and amplification protocol, were experimentally optimized for the Panther Fusion open access system. Plasmids, containing the ITS1 target, were used to assess linearity, amplification efficiency, and limit of detection (LoD), determined by probit regression.Clinical validation was conducted using residual lower respiratory tract samples from patients with suspected pulmonary fungal infection. Diagnostic performance was evaluated by comparing the assay with conventional microbiological methods, including culture and galactomannan testing. The evaluation was conducted in accordance with the guidelines to verify and validate a clinical microbiology test.This protocol enables laboratories to implement a reliable molecular tool for the detection of A. fumigatus within a fully automated platform, supporting early diagnosis of invasive pulmonary aspergillosis.

Keywords: Invasive-fungal infection; Laboratory-developed test (LDT); Molecular diagnosis; Panther Fusion; Pulmonary aspergillosis; Pulmonary infection; Real-time PCR.